Compositions comprising a fatty acid oil mixture and a free fatty acid, and methods and uses thereof

ABSTRACT

Compositions comprising a fatty acid oil mixture and at least one free fatty acid, and uses thereof are disclosed. Further disclosed are preconcentrates capable of forming a self- nanoemulsifying drug delivery system (SNEDDS), a self-microemulsifying drug delivery system (SMEDDS) or self-emulsifying drug delivery systems (SEDDS) in an aqueous solution. Preferred fatty acids are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in a form chosen from ethyl ester and triglyceride.

This application is a continuation of U.S. patent application Ser. No.15/350,522, filed Nov. 14, 2016, which is a division of U.S. patentapplication Ser. No. 13/255,602, filed May 3, 2012, which is a nationalstage application under 35 U.S.C. 371 of International PatentApplication No. PCT/IB2010/000788, filed Mar. 9, 2010, which claimspriority to U.S. Provisional Application No. 61/158,613, filed Mar. 9,2009, U.S. Provisional Application No. 61/242,630, filed Sep. 15, 2009,U.S. Provisional Application No. 61/254,291, filed Oct. 23, 2009, andU.S. Provisional Application No. 61/254,293, filed Oct. 23, 2009; all ofthe above-identified applications are incorporated herein by referencein their entireties.

The present disclosure relates generally to compositions comprising afatty acid oil mixture and at least one free fatty acid, and methods ofuse thereof. The fatty acid oil mixture may comprise omega-3 fattyacids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA) in ethyl ester or triglyceride form. Further disclosed arepreconcentrate compositions and self-nanoemulsifying drug deliverysystems (SNEDDS), self-microemulsifying drug delivery systems (SMEDDS)and self-emulsifying drug delivery systems (SEDDS).

The compositions presently disclosed may be administered, e.g., incapsule or tablet form, to a subject for therapeutic treatment and/orregulation of at least one health problem including, for example,irregular plasma lipid levels, cardiovascular functions, immunefunctions, visual functions, insulin action, neuronal development,hypertriglyceridemia, heart failure, and post myocardial infarction(MI). The present disclosure further relates to a method of increasinghydrolysis, solubility, bioavailability, absorption, and/or anycombination thereof.

In humans, cholesterol and triglycerides are part of lipoproteincomplexes in the bloodstream and can be separated viaultracentrifugation into high-density lipoprotein (HDL),intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL),and very-low-density lipoprotein (VLDL) fractions. Cholesterol andtriglycerides are synthesized in the liver, incorporated into VLDL, andreleased into the plasma. High levels of total cholesterol (total-C),LDL-C, and apolipoprotein B (a membrane complex for LDL-C and VLDL-C)promote human atherosclerosis and decreased levels of HDL-C and itstransport complex; apolipoprotein A is also associated with thedevelopment of atherosclerosis. Furthermore, cardiovascular morbidityand mortality in humans can vary directly with the level of total-C andLDL-C and inversely with the level of HDL-C. In addition, researchsuggests that non-HDL cholesterol is an indicator ofhypertriglyceridemia, vascular disease, atherosclerotic disease, andrelated conditions. In fact, NCEP ATP III specifies non-HDL cholesterolreduction as a treatment objective.

Omega-3 fatty acids may regulate plasma lipid levels, cardiovascular andimmune functions, insulin action, and neuronal development, and visualfunction. Marine oils, also commonly referred to as fish oils, are asource of omega-3 fatty acids, including eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), have been found to regulate lipidmetabolism. Plant-based oils and microbial oils are also sources ofomega-3 fatty acids. Omega-3 fatty acids may have beneficial effects onthe risk factors for cardiovascular diseases, for example hypertensionand hypertriglyceridemia, and on the coagulation factor VII phospholipidcomplex activity. Omega-3 fatty acids may also lower serumtriglycerides, increase serum HDL cholesterol, lower systolic anddiastolic blood pressure and/or pulse rate, and may lower the activityof the blood coagulation factor VII-phospholipid complex. Further,omega-3 fatty acids are generally well-tolerated, without giving rise tosevere side effects.

Several formulations of omega-3 fatty acids have been developed. Forexample, one form of omega-3 fatty acid oil mixture is a concentrate ofprimary omega-3, long chain, polyunsaturated fatty acids from fish oilcontaining DHA and EPA, such as sold under the trademarkOmacor®/Lovaza™/Zodin®/Seacor®. See, for example, U.S. Pat. Nos.5,502,077, 5,656,667 and 5,698,594. In particular, each 1000 mg capsuleof Lovaza™ contains at least 90% omega-3 ethyl ester fatty acids (84%EPA/DHA); approximately 465 mg EPA ethyl ester and approximately 375 mgDHA ethyl ester.

Further, for example, EPA/DHA ethyl esters have also been used incompositions for delivery of therapeutic drugs. For instance, U.S. Pat.No. 6,284,268 (Cyclosporine Therapeutics Ltd.) describes aself-emulsifying microemulsion or emulsion preconcentrate pharmaceuticalcompositions containing an omega-3 fatty acid oil and poorly watersoluble therapeutic agent such as cyclosporine for oral administration.Cyclosporines are claimed to have additive or synergistic therapeuticeffects with omega-3 fatty acid oil. The '268 patent discloses greatersolubility and stability of cyclosporine formulations comprising omega-3fatty acid oils. WO 99/29300 (RTP Pharma) relates to self-emulsifyingfenofibrate formulations based on a hydrophobic component selected fromtriglyceride, diglyceride, monoglycerides, free fatty acids and fattyacids and derivatives thereof.

However, evidence suggests that long chain fatty acids and alcohols ofup to at least C₂₄ are reversibly interconverted. Enzyme systems existin the liver, fibroblasts, and the brain that convert fatty alcohols tofatty acids. In some tissues, fatty acids can be reduced back toalcohols. The carboxylic acid functional group of fatty acid moleculestargets binding, but this ionizable group may hinder the molecule fromcrossing the cell membranes, such as of the intestinal wall. As aresult, carboxylic acid functional groups are often protected as esters.The ester is less polar than the carboxylic acid, and may more easilycross the fatty cell membranes. Once in the bloodstream, the ester canbe hydrolyzed back to the free carboxylic acid by enzyme esterase in theblood. It may be possible that the plasma enzymes do not hydrolyze theester fast enough, however, and that the conversion of ester to freecarboxylic acid predominantly takes place in the liver. Ethyl esters ofpolyunsaturated fatty can also be hydrolyzed to free carboxylic acids invivo.

Thus, there remains a need in the art for compositions and/or methodsthat improve or enhance solubilization, digestion, bioavailabilityand/or absorption of omega-3 fatty acids in vivo, while maintaining theability to cross cell membranes.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the present disclosure, as claimed.

The present disclosure is directed to a pharmaceutical compositioncomprising: a fatty acid oil mixture comprising at least 75%eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), by weight ofthe fatty acid oil mixture, wherein the EPA and DHA are in a form chosenfrom ethyl ester and triglyceride; and at least one free fatty acid.

The present disclosure is also directed to a pharmaceuticalpreconcentrate comprising: a fatty acid oil mixture comprising at least75% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), byweight of the fatty acid oil mixture, wherein the EPA and DHA are in aform chosen from ethyl ester and triglyceride; at least one free fattyacid; and at least one surfactant.

For example, the present disclosure provides for a pharmaceuticalpreconcentrate comprising: a fatty acid oil mixture comprising fromabout 80% to about 88% eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA), by weight of the fatty acid oil mixture, wherein the EPA andDHA are in ethyl ester form; at least one free fatty acid comprisingfrom about 80% to about 88% eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), by weight of the at least one free fattyacid, wherein the EPA and DHA are in free fatty acid form; and at leastone surfactant chosen from polysorbate 20, polysorbate 80, and mixturesthereof.

Further for example, the present disclosure provides for apharmaceutical preconcentrate comprising: from about 45% to about 55% byweight, relative to the weight of the preconcentrate, of a fatty acidoil mixture comprising from about 80% to about 88% eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) by weight of the fatty acid oilmixture, wherein the EPA and DHA are in a form chosen from ethyl esterand triglyceride; from about 5% to about 15% of at least one free fattyacid, by weight relative to the weight of the preconcentrate; and fromabout 30% to about 40% of at least one surfactant, by weight relative tothe weight of the preconcentrate.

The present disclosure is also directed to a pharmaceuticalpreconcentrate comprising: from about 55% to about 65% by weight,relative to the weight of the preconcentrate, of a fatty acid oilmixture comprising from about 80% to about 88% eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) by weight of the fatty acid oilmixture, wherein the EPA and DHA are in a form chosen from ethyl esterand triglyceride; from about 5% to about 15% of at least one free fattyacid, by weight relative to the weight of the preconcentrate; and fromabout 20% to about 30% of at least one surfactant, by weight relative tothe weight of the preconcentrate.

The present disclosure is yet still further directed to a pharmaceuticalpreconcentrate comprising: a fatty acid oil mixture comprising fromabout 80% to about 88% eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA) by weight of the fatty acid oil mixture, wherein the EPA andDHA are in ethyl ester form; at least one free fatty acid comprisingoleic acid; and at least one surfactant chosen from polysorbate 20 andpolysorbate 80.

The present disclosure is also directed to a self-nanoemulsifying drugdelivery system (SNEDDS), self-microemulsifying drug delivery system(SMEDDS), or self-emulsifying drug delivery system (SEDDS) comprising apharmaceutical preconcentrate comprising: a fatty acid oil mixturecomprising at least 75% eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA), by weight of the fatty acid oil mixture, wherein the EPA andDHA are in a form chosen from ethyl ester and triglyceride; at least onefree fatty acid; and at least one surfactant; wherein the preconcentrateforms an emulsion in an aqueous solution.

The present disclosure further provides for a method of treating atleast one health problem in a subject in need thereof comprisingadministering to the subject a pharmaceutical composition comprising: apharmaceutically-effective amount of a fatty acid oil mixture comprisingat least 75% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA),by weight of the fatty acid oil mixture, wherein the EPA and DHA are ina form chosen from ethyl ester and triglyceride; and at least one freefatty acid; wherein the at least one health problem is chosen fromirregular plasma lipid levels, cardiovascular functions, immunefunctions, visual functions, insulin action, neuronal development, heartfailure, and post myocardial infarction. For example, the compositionfurther comprises at least one surfactant to form a pharmaceuticalpreconcentrate, such as, the preconcentrate forms a self-nanoemulsifyingdrug delivery system (SNEDDS), self-microemulsifying drug deliverysystem (SMEDDS), or self-emulsifying drug delivery system (SEDDS) in anaqueous solution.

In addition, the present disclosure is directed to a food supplement ornutritional supplement composition comprising: a fatty acid oil mixturecomprising from about 25% to about 75% eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), by weight of the fatty acid oil mixture,wherein the EPA and DHA are in a form chosen from ethyl ester andtriglyceride; and at least one free fatty acid. For example, thecomposition further comprises at least one surfactant to form asupplement preconcentrate, such as the preconcentrate forms aself-nanoemulsifying drug delivery system (SNEDDS),self-microemulsifying drug delivery system (SMEDDS), or self-emulsifyingdrug delivery system (SEDDS) in an aqueous solution.

The present disclosure is also directed to a method for enhancing atleast one parameter chosen from hydrolysis, solubility, bioavailability,absorption, and combinations thereof of eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) comprising combining: a fatty acid oilmixture comprising EPA and DHA in a form chosen from ethyl ester andtriglyceride; and at least one free fatty acid. For example, a methodfor enhancing at least one parameter chosen from hydrolysis, solubility,bioavailability, absorption, and combinations thereof ofeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) comprisingcombining: a fatty acid oil mixture comprising EPA and DHA in a formchosen from ethyl ester and triglyceride; at least one free fatty acid;and at least one surfactant; wherein the fatty acid oil mixture, that atleast one free fatty acid, and the at least one surfactant form apreconcentrate. In addition, the preconcentrate forms aself-nanoemulsifying drug delivery system (SNEDDS),self-microemulsifying drug delivery system (SMEDDS), or self-emulsifyingdrug delivery system (SEDDS) in an aqueous solution.

In a further embodiment, the present disclosure is directed to apharmaceutical composition comprising a fatty acid oil mixturecomprising at least 75% eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA), by weight of the fatty acid oil mixture, wherein the EPA andDHA are in a form chosen from ethyl ester and triglyceride; and at leastone free fatty acid for the treatment of at least one health problemchosen from irregular plasma lipid levels, cardiovascular functions,immune functions, visual functions, insulin action, neuronaldevelopment, heart failure, and post myocardial infarction.

In yet still a further embodiment, the present disclosure provides for apharmaceutical preconcentrate comprising a fatty acid oil mixturecomprising at least 75% eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA), by weight of the fatty acid oil mixture, wherein the EPA andDHA are in a form chosen from ethyl ester and triglyceride; at least onefree fatty acid; and at least one surfactant for the treatment of atleast one health problem chosen from irregular plasma lipid levels,cardiovascular functions, immune functions, visual functions, insulinaction, neuronal development, heart failure, and post myocardialinfarction.

The present disclosure is also directed to a self-nanoemulsifying drugdelivery system (SNEDDS), self-microemulsifying drug delivery system(SMEDDS), or self-emulsifying drug delivery system (SEDDS) comprising apharmaceutical preconcentrate comprising: a fatty acid oil mixturecomprising at least 75% eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA), by weight of the fatty acid oil mixture, wherein the EPA andDHA are in a form chosen from ethyl ester and triglyceride; at least onefree fatty acid; and at least one surfactant; wherein the preconcentrateforms an emulsion in an aqueous solution for the treatment of at leastone health problem chosen from irregular plasma lipid levels,cardiovascular functions, immune functions, visual functions, insulinaction, neuronal development, heart failure, and post myocardialinfarction.

The present disclosure is further directed to a method of regulating atleast one health problem in a subject in need thereof comprisingadministering to the subject a supplement composition comprising: afatty acid oil mixture comprising from about 25% to about 75%eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), by weight ofthe fatty acid oil mixture, wherein the EPA and DHA are in a form chosenfrom ethyl ester and triglyceride; and at least one free fatty acid;wherein the at least one health problem is chosen from irregular plasmalipid levels, cardiovascular functions, immune functions, visualfunctions, insulin action, neuronal development, heart failure, and postmyocardial infarction.

The present disclosure is also further directed to a method ofregulating at least one health problem in a subject in need thereofcomprising administering to the subject a supplement compositioncomprising: a fatty acid oil mixture comprising from about 25% to about75% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), byweight of the fatty acid oil mixture, wherein the EPA and DHA are in aform chosen from ethyl ester and triglyceride; at least one free fattyacid; and at least one surfactant; wherein the at least one healthproblem is chosen from irregular plasma lipid levels, cardiovascularfunctions, immune functions, visual functions, insulin action, neuronaldevelopment, heart failure, and post myocardial infarction.

The present disclosure is yet still further directed to a foodsupplement or nutritional supplement composition comprising: a fattyacid oil mixture comprising from about 25% to about 75% eicosapentaenoicacid (EPA) and docosahexaenoic acid (DHA), by weight of the fatty acidoil mixture, wherein the EPA and DHA are in a form chosen from ethylester and triglyceride; and at least one free fatty acid for theregulation of at least one health problem chosen from irregular plasmalipid levels, cardiovascular functions, immune functions, visualfunctions, insulin action, neuronal development, heart failure, and postmyocardial infarction.

The present disclosure is also further directed to a food supplement ornutritional supplement preconcentrate comprising: a fatty acid oilmixture comprising from about 25% to about 75% eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA), by weight of the fatty acid oilmixture, wherein the EPA and DHA are in a form chosen from ethyl esterand triglyceride; at least one free fatty acid; and at least onesurfactant for the regulation of at least one health problem chosen fromirregular plasma lipid levels, cardiovascular functions, immunefunctions, visual functions, insulin action, neuronal development, heartfailure, and post myocardial infarction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the viscosity of preconcentrates A-L.

FIG. 2 shows the average particle size distribution for preconcentratesA-F, I, and J in gastric media and intestinal media.

FIG. 3 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate A in gastric media.

FIG. 4 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate B in gastric media.

FIG. 5 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate C in gastric media.

FIG. 6 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate D in gastric media.

FIG. 7 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate E in gastric media.

FIG. 8 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate F in gastric media.

FIG. 9 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate I in gastric media.

FIG. 10 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate J in gastric media.

FIG. 11 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate A in intestinal media.

FIG. 12 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate B in intestinal media.

FIG. 13 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate C in intestinal media.

FIG. 14 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate D in intestinal media.

FIG. 15 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate E in intestinal media.

FIG. 16 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate F in intestinal media.

FIG. 17 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate I in intestinal media.

FIG. 18 shows the read out from the Malvern zetasizer for fourconsecutive measurements on preconcentrate J in intestinal media.

FIG. 19 shows the disappearance of EPA-EE and DHA-EE and the appearanceof EPA-FA and DHA-FA during lipolysis of Omacor®.

FIG. 20 shows the percent recovery of EPA+DHA at different time-pointsfor Omacor®.

FIG. 21 shows the percent lipolysis of EPA-EE, DHA-EE and total K85EE atdifferent time points for Omacor®.

FIG. 22 shows the disappearance of EPA-EE and DHA-EE and the appearanceof EPA-FA and DHA-FA during lipolysis of preconcentrate A.

FIG. 23 shows the percent recovery of EPA+DHA at different time-pointsfor preconcentrate A.

FIG. 24 shows the percent lipolysis of EPA-EE, DHA-EE and total K85EE atdifferent time points for preconcentrate A.

FIG. 25 shows the disappearance of EPA-EE and DHArEE and the appearanceof EPA-FA and DHA-FA during lipolysis of preconcentrate B.

FIG. 26 shows the percent recovery of EPA+DHA at different time-pointsfor preconcentrate B.

FIG. 27 shows the percent lipolysis of EPA-EE, DHA-EE and total K85EE atdifferent time points for preconcentrate B.

FIG. 28 shows the disappearance of EPA-EE and DHA-EE and the appearanceof EPA-FA and DHA-FA during lipolysis of preconcentrate C.

FIG. 29 shows the percent recovery of EPA+DHA at different time-pointsfor preconcentrate C.

FIG. 30 shows the percent lipolysis of EPA-EE, DHA-EE and total K85EE atdifferent time points for preconcentrate C.

FIG. 31 shows the disappearance of EPA-EE and DHA-EE and the appearanceof EPA-FA and DHA-FA during lipolysis of preconcentrate D.

FIG. 32 shows the percent recovery of EPA+DHA at different time-pointsfor preconcentrate D.

FIG. 33 shows the percent lipolysis of EPA-EE, DHA-EE and total K85EE atdifferent time points for preconcentrate D.

FIG. 34 shows the disappearance of EPA-EE and DHA-EE and the appearanceof EPA-FA and DHA-FA during lipolysis of preconcentrate E.

FIG. 35 shows the percent recovery of EPA+DHA at different time-pointsfor preconcentrate E.

FIG. 36 shows the percent lipolysis of EPA-EE, DHA-EE and total K85EE atdifferent time points for preconcentrate E.

FIG. 37 shows the plasma concentration versus time profile of the totallipid concentration of EPA for Example 14.

DESCRIPTION

Particular aspects of the disclosure are described in greater detailbelow. The terms and definitions as used in the present application andas clarified herein are intended to represent the meaning within thepresent disclosure. The patent and scientific literature referred toherein and referenced above is hereby incorporated by reference. Theterms and definitions provided herein control, if in conflict with termsand/or definitions incorporated by reference.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context dictates otherwise.

The terms “approximately” and “about” mean to be nearly the same as areferenced number or value. As used herein, the terms “approximately”and “about” should be generally understood to encompass ±10% of aspecified amount, frequency or value.

The terms “administer,” “administration” or “administering” as usedherein refer to (1) providing, giving, dosing and/or prescribing byeither a health practitioner or his authorized agent or under hisdirection a composition according to the disclosure, and (2) puttinginto, taking or consuming by the patient or person himself or herself, acomposition according to the disclosure.

The present disclosure provides for pharmaceutical and supplementcompositions comprising a fatty acid oil mixture and at least one freefatty acid, and methods of use thereof. The compositions can furthercomprise at least one surfactant to form a preconcentrate. Thepreconcentrates of the present disclosure can produce dispersions of lowor very low mean particle size when mixed with an aqueous medium. Suchdispersions can be characterized as nanoemulsions, microemulsions, oremulsions. For example, upon delivery, the preconcentrates are thoughtto produce dispersions with gastric or other physiological fluidsgenerating self-nanoemulsifying drug delivery systems (SNEDDS),self-microemulsifying drug delivery systems (SMEDDS), or selfemulsifying drug delivery systems (SEDDS).

Fatty Acid Oil Mixture

Compositions of the present disclosure comprise at least one fatty acidoil mixture. The fatty acid oil mixture comprises eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA). As used herein, the term “fattyacid oil mixture” includes fatty acids, such as unsaturated (e.g.,monounsaturated, polyunsaturated) or saturated fatty acids, as well aspharmaceutically-acceptable esters, free acids, mono-, di- andtriglycerides, derivatives, conjugates, precursors, salts, and mixturesthereof. In at least one embodiment, the fatty acid oil mixturecomprises fatty acids, such as omega-3 fatty acids, in a form chosenfrom ethyl ester and triglyceride.

The term “omega-3 fatty acids” includes natural and synthetic omega-3fatty acids, as well as pharmaceutically-acceptable esters, free acids,triglycerides, derivatives, conjugates (see, e.g., Zaloga et al., U.S.Patent Application Publication No. 2004/0254357, and Horrobin et al.,U.S. Pat. No. 6,245,811, each hereby incorporated by reference),precursors, salts, and mixtures thereof. Examples of omega-3 fatty acidoils include, but are not limited to, omega-3 polyunsaturated,long-chain fatty acids such as eicosapentaenoic acid (EPA),docosahexaenoic acid (DHA), α-linolenic acid (ALA), heneicosapentaenoicacid (HPA), docosapentaenoic acid (DPA), eicosatetraenoic acid (ETA),eicosatrienoic acid (ETE), and octadecatetraenoic acid (i.e.,stearidonic acid, STA); esters of omega-3 fatty acids with glycerol suchas mono-, di- and triglycerides; and esters of the omega-3 fatty acidsand a primary, secondary and/or tertiary alcohol, such as, for example,fatty acid methyl esters and fatty acid ethyl esters. The omega-3 fattyacids, esters, triglycerides, derivatives, conjugates, precursors, saltsand/or mixtures thereof according to the present disclosure can be usedin their pure form and/or as a component of an oil, for example, asmarine oil (e.g., fish oil and purified fish oil concentrates), algaeoils, microbial oils and plant-based oils.

In some embodiments of the present disclosure, the fatty acid oilmixture comprises EPA and DHA. Further for example, the fatty acid oilmixture comprises EPA and DHA in a form chosen from ethyl ester andtriglyceride.

The fatty acid oil mixture of the present disclosure may furthercomprise at least one fatty acid other than EPA and DHA. Examples ofsuch fatty acids include, but are not limited to, omega-3 fatty acidsother than EPA and DHA and omega-6 fatty acids. For example, in someembodiments of the present disclosure, the fatty acid oil mixturecomprises at least one fatty acid other than EPA and DHA chosen fromα-linolenic acid (ALA), heneicosapentaenoic acid (HPA), docosapentaenoicacid (DPA), eicosatetraenoic acid (ETA), eicosatrienoic acid (ETE), andstearidonic acid (STA). In some embodiments, the at least one fatty acidother than EPA and DHA is chosen from linoleic acid, gamma-linolenicacid (GLA), arachidonic acid (AA), docosapentaenoic acid (i.e., osbondacid), and mixtures thereof. In some embodiments, the at least one fattyacid other than EPA and DHA is in a form chosen from ethyl ester andtriglyceride.

Examples of further fatty acids, or mixtures thereof (fatty acid oilmixtures) encompassed by the present disclosure include, but are notlimited to, the fatty acids defined in the European PharamacopoeiaOmega-3 Ethyl Esters 90 and purified marine oils, for example, theEuropean Pharamacopoeia Omega-3 Acid Triglycerides, the EuropeanPharamacopoeia Omega-3 acid Ethyl Esters 60, the European PharmacopoeiaFish Oil Rich in Omega-3 Acids monograph, and/or for instance, the USPfish oil monograph.

Commercial examples of fatty acid oil mixtures comprising differentfatty acids suitable for the present disclosure include, but are notlimited to: Incromega™ omega-3 marine oil concentrates such asIncromega™ TG7010 SR, Incromega™ E7010 SR, Incromega™ TG6015, Incromega™EPA500TG SR, Incromega™ E400200 SR, Incromega™ E4010, Incromega™DHA700TG SR, Incromega™ DHA700E SR, Incromega™ DHA500TG SR, Incromega™TG3322 SR, Incromega™ E3322 SR, Incromega™ TG3322, Incromega™ E3322,Incromega™ Trio TG/EE (Croda International PLC, Yorkshire, England);EPAX6000FA, EPAX5000TG, EPAX4510TG, EPAX2050TG, EPAX7010EE, EPAX5500EE,EPAX5500TG, EPAX5000EE, EPAX5000TG, EPAX6000EE, EPAX6000TG, EPAX6000FA,EPAX6500EE, EPAX6500TG, EPAX4510TG, EPAX1050TG, EPAX2050TG, EPAX 7010TG,EPAX7010EE, EPAX6015TG/EE, EPAX4020TG, and EPAX4020EE (EPAX is awholly-owned subsidiary of Norwegian company Austevoll Seafood ASA);Omacor®/Lovaza™/Zodin®/Seacor® finished pharmaceutical product, K85EE,and AGP 103 (Pronova BioPharma Norge AS); MEG-3® EPA/DHA fish oilconcentrates (Ocean Nutrition Canada); DHA FNO “Functional NutritionalOil” and DHA CL “Clear Liquid” (Lonza); Superba™ Krill Oil (Aker);omega-3 products comprising DHA produced by Martek; Neptune krill oil(Neptune); cod-liver oil products and anti-reflux fish oil concentrate(TG) produced by Møllers; Lysi Omega-3 Fish oil; Seven Seas Triomega®Cod Liver Oil Blend (Seven Seas); Fri Flyt Omega-3 (Vesteralens); andEpadel (Mochida). Those commercial embodiments provide for variousomega-3 fatty acids, combinations, and other components as a result ofthe transesterification process or method of preparation in order toobtain the omega-3 fatty acid(s) from various sources, such as marine,algae, microbial, and plant-based sources.

In some embodiments of the present disclosure, the fatty acid oilmixture comprises at least one fatty acid derivative, such as analpha-substituted omega-3 fatty acid derivative. The at least one alphasubstituted omega-3 fatty acid derivative may be substituted, forexample, at the second carbon atom from the functional group of theomega-3 fatty acid with at least one substituent chosen from hydrogen,hydroxyl groups, alkyl groups, such as C₁-C₃ alkyl groups, and alkoxygroups. In one embodiment of the present disclosure, the at least onealpha-substituted omega-3 fatty acid derivative is chosen frommono-substituted and di-substituted fatty acids. In one embodiment, theat least one alpha substituted omega-3 fatty acid derivative is chosenfrom alpha-substituted C₁₄-C₂₄ alkenes having 2 to 6 double bonds. Inanother embodiment, the at least one alpha-substituted omega-3 fattyacid derivative is chosen from alpha-substituted C₁₄-C₂₄ alkenes having5 or 6 double bonds in cis configuration.

In some embodiments, the fatty acid oil mixture comprises EPA and/or DHAin a form of an alpha-substituted fatty acid derivative. For example, inone embodiment, the fatty acid oil mixture comprises EPA in a form of analpha-substituted derivative. In another embodiment, the fatty acid oilmixture comprises DHA in a form of an alpha-substituted derivative. Inyet another embodiment, the fatty acid oil mixture comprises EPA and DHAin a form of an alpha-substituted derivative.

In some embodiments, the fatty acid oil mixture comprises EPA and DHA,and further comprises at least one alpha-substituted omega-3 fatty acidderivative. For example, in some embodiments, the fatty acid oil mixturecomprises EPA and DHA, and at least one of EPA and DHA in a form of analpha-substituted derivative.

In another embodiment, the EPA and DHA of the fatty acid oil mixture isat least one alpha-substituted omega-3 fatty acid derivative.

The fatty acid oil mixture according to the present disclosure may bederived from animal oils and/or non-animal oils. In some embodiments ofthe present disclosure, the fatty acid oil mixture is derived from atleast one oil chosen from marine oil, algae oil, plant-based oil, andmicrobial oil. Marine oils include, for example, fish oil, krill oil,and lipid composition derived from fish. Plant-based oils include, forexample, flaxseed oil, canola oil, mustard seed oil, and soybean oil.Microbial oils include, for example, products by Martek. In at least oneembodiment of the present disclosure, the fatty acid oil mixture isderived from a marine oil, such as a fish oil. In at least oneembodiment, the marine oil is a purified fish oil.

In some embodiments of the present disclosure, the fatty acids, such asomega-3 fatty acids, of the fatty acid oil mixture are esterified, suchas alkyl esters and further for example, ethyl ester. In otherembodiments, the fatty acids are chosen from mono-, di-, andtriglycerides.

In some embodiments, the fatty acid oil mixture is obtained by atransesterification of the body oil of a fat fish species coming from,for example, anchovy or tuna oil, and subsequent physico-chemicalpurification processes, including urea fractionation followed bymolecular distillation. In some embodiments, the crude oil mixture mayalso be subjected to a stripping process for decreasing the amount ofenvironmental pollutants and/or cholesterol before thetransesterification.

In another embodiment, the fatty acid oil mixture is obtained by usingsupercritical CO₂ extraction or chromatography techniques, for exampleto up-concentrate primary EPA and DHA from fish oil concentrates.

In some embodiments of the present disclosure, at least one of theomega-3 fatty acids of the fatty acid oil mixture has a cisconfiguration. Examples include, but are not limited to,(all-Z)-9,12,15-octadecatrienoic acid (ALA),(all-Z)-6,9,12,15-octadecatetraenoic acid (STA),(all-Z)-11,14,17-eicosatrienoic acid (ETE),(all-Z)-5,8,11,14,17-eicosapentaenoic acid (EPA),(all-Z)-4,7,10,13,16,19-docosahexaenoic acid (DHA),(all-Z)-8,11,14,17-eicosatetraenoic acid (ETA),(all-Z)-7,10,13,16,19-docosapentaenoic acid (DPA),(all-Z)-6,9,12,15,19-heneicosapentaenoic acid (HPA);(all-Z)-5,8,11,14-eicosatetraenoic acid,(all-Z)-4,7,10,13,16-docosapentaenoic acid (osbond acid),(all-Z)-9,12-octadecadienoic acid (linoleic acid),(all-Z)-5,8,11,14-eicosatetraenoic acid (AA),(all-Z)-6,9,12-octadecatrienoic acid (GLA); (Z)-9-octadecenoic acid(oleic acid), 13(Z)-docosenoic acid (erucic acid),(R-(Z))-12-hydroxy-9-octadecenoic acid (ricinoleic acid).

In some embodiments of the present disclosure, the weight ratio ofEPA:DHA of the fatty acid oil mixture ranges from about 1:10 to about10:1, from about 1:8 to about 8:1, from about 1:6 to about 6:1, fromabout 1:5 to about 5:1, from about 1:4 to about 4:1, from about 1:3 toabout 3:1, or from about 1:2 to 2 about :1. In at least one embodiment,the weight ratio of EPA:DHA of the fatty acid oil mixture ranges fromabout 1:2 to about 2:1. In at least one embodiment, the weight ratio ofEPA:DHA of the fatty acid oil mixture ranges from about 1:1 to about2:1. In at least one embodiment, the weight ratio of EPA:DHA of thefatty acid oil mixture ranges from about 1.2 to about 1.3.

Free Fatty Acid (FFA)

The compositions presently disclosed comprise at least one free fattyacid. Without being bound by theory, it is believed that the addition ofat least one free fatty acid may enhance or improve lipolysis of thefatty acid oil mixture in the body, e.g., the interconversion of fattyacid esters and/or triglycerides to the free fatty acid form forefficient uptake. The addition of at least one free fatty acid may, forexample, provide for enhanced or improved hydrolysis, solubility,bioavailability, absorption, or any combinations thereof of fatty acidsof the fatty acid oil mixture in vivo.

Examples of free fatty acids include, but are not limited to, EPA, DHA,α-linolenic acid (ALA), heneicosapentaenoic acid (HPA), docosapentaenoicacid (DPA), eicosatetraenoic acid (ETA), eicosatrienoic acid (ETE),stearidonic acid (STA), linoleic acid, gamma-linolenic acid (GLA),arachidonic acid (AA), osbond acid, oleic acid, ricinoleic acid, erucicacid, and mixtures thereof. In at least one embodiment, the at least onefree fatty acid is a polyunsaturated fatty acid.

In some embodiments, the at least one free fatty acid is chosen fromoleic acid, ricinoleic acid, linoleic acid, and erucic acid. In oneembodiment, the at least one free fatty acid comprises oleic acid orlinoleic acid.

In some embodiments, the at least one free fatty acid comprises at least80% omega-3 fatty acids by weight of the at least one free fatty acid,such as at least 90% omega-3 fatty acids by weight of the at least onefree fatty acid.

In some embodiments, the at least one free fatty acid comprises at least75% EPA and DHA by weight of the at least one free fatty acid. Forexample, in some embodiments, the at least one free fatty acid comprisesat least 80% by weight, at least 85% by weight, at least 90% by weight,or at least 95% EPA and DHA, by weight of the at least one free fattyacid. In some embodiments, the at least one free fatty acid comprisesabout 80% EPA and DHA by weight of the at least one free fatty acid,such as about 85%, about 90%, about 95%, or any number in between, byweight of the at least one free fatty acid. The at least one free fattyacid can be used in a pure form and/or as a component of an oil, forexample, as marine oil (e.g., fish oil and purified fish oilconcentrates), microbial oil and plant-based oils.

In some embodiments, the at least one free fatty acid comprises fromabout 75% to about 95% EPA and DHA by weight of the at least one freefatty acid, such as from about 75% to about 90%, from about 75% to about85%, from about 75% to about 80%, from about 80% to about 95%, fromabout 80% to about 90%, from about 80% to about 85%, from about 85% toabout 95%, from about 85% to about 90%, and further for example, fromabout 90% to about 95% by weight of the at least one free fatty acid, orany number in between. In at least one embodiment, the at least one freefatty acid comprises from about 80% to about 85% EPA and DHA, by weightof the at least one free fatty acid, such as from about 80% to about 88%EPA and DHA by weight, such as about 84%, by weight of the at least onefree fatty acid.

Commercial embodiments of at least one free fatty acid encompassed bythe present disclosure include, but are not limited to, K85FA (PronovaBioPharma Norge AS).

Pharmaceutical

In some embodiments of the present disclosure, the fatty acid oilmixture acts as an active pharmaceutical ingredient (API). For example,the present disclosure provides for a pharmaceutical compositioncomprising a fatty acid oil mixture and at least one free fatty acid. Insome embodiments, the fatty acid oil mixture is present in apharmaceutically-acceptable amount. As used herein, the term“pharmaceutically-effective amount” means an amount sufficient to treat,e.g., reduce and/or alleviate the effects, symptoms, etc., at least onehealth problem in a subject in need thereof. In at least someembodiments of the present disclosure, the fatty acid oil mixture doesnot comprise an additional active agent.

Where the composition is a pharmaceutical composition, the fatty acidoil mixture comprises at least 75% EPA and DHA by weight of the fattyacid oil mixture. For example, in one embodiment, the fatty acid oilmixture comprises at least 80% EPA and DHA by weight of the fatty acidoil mixture, such as at least 85%, at least 90%, or at least 95%, byweight of the fatty acid oil mixture. In some embodiments, the fattyacid oil mixture comprises about 80% EPA and DHA by weight of the fattyacid oil mixture, such as about 85%, about 90%, about 95%, or any numberin between, by weight of the fatty acid oil mixture.

For example, in some embodiments, the fatty acid oil mixture comprisesfrom about 75% to about 95% EPA and DHA by weight of the fatty acid oilmixture, such as from about 75% to about 90%, from about 75% to about88%, from about 75% to about 85%, from about 75% to about 80%, fromabout 80% to about 95%, from about 80% to about 90%, from about 80% toabout 85%, from about 85% to about 95%, from about 85% to about 90%, andfurther for example, from about 90% to about 95% EPA and DHA, by weightof the fatty acid oil mixture, or any number in between. In at least oneembodiment, the fatty acid oil mixture comprises from about 80% to about85% EPA and DHA, by weight of the fatty acid oil mixture, such as fromabout 80% to about 88%, such as about 84%, by weight of the fatty acidoil mixture.

In some embodiments, the fatty acid oil mixture comprises at least 95%of EPA or DHA, or EPA and DHA, by weight of the fatty acid oil mixture,wherein the EPA and DHA are in ethyl ester form.

In a further embodiment, the fatty acid oil mixture may comprise otheromega-3 fatty acids. For example, the present disclosure encompasses atleast 90% omega-3 fatty acids, by weight of the fatty acid oil mixture.

In one embodiment, for example, the fatty acid oil mixture comprisesfrom about 75% to about 88% EPA and DHA, by weight of the fatty acid oilmixture, wherein the EPA and DHA are in ethyl ester form; wherein thefatty acid oil mixture comprises at least 90% of omega-3 fatty acids inethyl ester form, by weight of the fatty acid oil mixture.

In another embodiment, the fatty acid oil mixture comprises from about75% to about 88% EPA and DHA, by weight of the fatty acid oil mixture,wherein the EPA and DHA are in ethyl ester form; wherein the fatty acidoil mixture comprises at least 90% of omega-3 fatty acids in ethyl esterform, by weight of the fatty acid oil mixture, and wherein the fattyacid oil mixture comprises a-linolenic acid (ALA).

In one embodiment, the fatty acid oil mixture comprises from about 80%to about 88% EPA and DHA by weight of the fatty acid oil mixture,wherein the EPA and DHA are in ethyl ester form, and further comprisesdocosapentaenoic acid (DPA) in ethyl ester form.

In another embodiment, the fatty acid oil mixture comprises from about80% to about 88% EPA and DHA by weight of the fatty acid oil mixture,wherein the EPA and DHA are in ethyl ester form, and further comprisesfrom about 1% to about 4% (all-Zomega-3)-6,9,12,15,18-heneicosapentaenoic acid (HPA) in ethyl esterform, by weight of the fatty acid oil mixture.

In another embodiment, the fatty acid oil mixture comprises from about80% to about 88% EPA and DHA by weight of the fatty acid oil mixture,wherein the EPA and DHA are in ethyl ester form; and from 1% to about 4%fatty acid ethyl esters other than EPA and DHA, by weight of the fattyacid oil mixture, wherein the fatty acid ethyl esters other than EPA andDHA have C₂₀, C₂₁, or C₂₂ carbon atoms.

In one embodiment, the fatty acid oil mixture may comprise K85EE or AGP103 (Pronova BioPharma Norge AS). In another embodiment, the fatty acidoil mixture may comprise K85TG (Pronova BioPharma Norge AS).

In one embodiment, the pharmaceutical composition comprising at leastK85EE, K85-FA, and Tween 20 or 80, for example, provide for enhancedbioavailability. For example, the bioavailability may be increased>about40%, such as, about 80%.

EPA and DHA Products

In at least one embodiment, the fatty acid oil mixture comprises atleast 75% EPA and DHA by weight of the fatty acid oil mixture, of whichat least 95% is EPA. In another embodiment, the fatty acid oil mixturecomprises at least 80% EPA and DHA by weight of the fatty acid oilmixture, of which at least 95% is EPA. In yet another embodiment, thefatty acid oil mixture comprises at least 90% EPA and DHA by weight ofthe fatty acid oil mixture, of which at least 95% is EPA.

In another embodiment, the fatty acid oil mixture comprises at least 75%EPA and DHA by weight of the fatty acid oil mixture, of which at least95% is DHA. For example, in one embodiment, the fatty acid oil mixturecomprises at least 80% EPA and DHA by weight of the fatty acid oilmixture, of which at least 95% is DHA. In another embodiment, the fattyacid oil mixture comprises at least 90% EPA and DHA by weight of thefatty acid oil mixture, of which at least 95% is DHA.

Supplement

The present disclosure further provides a food supplement or anutritional supplement comprising a fatty acid oil mixture and at leastone fatty acid, wherein the fatty acid oil mixture comprises less than75% EPA and DHA by weight of the fatty acid oil mixture. In someembodiments, for example, the fatty acid oil comprises less than 70% EPAand DHA by weight of the fatty acid oil mixture, such as less than 65%,less than 60%, less than 55%, less than 50%, less than 45%, less than40%, or even less than 35% by weight of the fatty acid oil mixture.

In some embodiments, the fatty acid oil mixture comprises from about 25%to about 75% EPA and DHA by weight of the fatty acid oil mixture, suchas from about 30% to about 75%, from about 30% to about 70%, from about30% to about 65%, from about 30% to about 55%, from about 30% to about50%, from about 30% to about 45%, from about 30% to about 40%, andfurther for example, from about 30% to about 35% EPA and DHA, by weightof the fatty acid oil mixture.

In some embodiments of the present disclosure, the fatty acids, such asomega-3 fatty acids, of the fatty acid oil mixture are esterified, suchas alkyl esters. The alkyl esters may include, but are not limited to,ethyl, methyl, propyl, and butyl esters, and mixtures thereof. In otherembodiments, the fatty acids are chosen from mono-, di-, andtriglycerides. For example, the fatty acid oil mixture comprises fromabout 25% to about 75% EPA and DHA, by weight of the fatty acid oilmixture in a form chosen from methyl ester, ethyl ester, andtriglyceride.

Compositions

In some embodiments, the fatty acid oil mixture comprises from about 50%to about 95% by weight and the at least one free fatty acid comprisesfrom about 5% to about 50% by weight, each relative to the total weightof the composition.

The compositions presently disclosed may be in a tablet form or in acapsule form.

Superdisintegrant

The compositions presently disclosed may further comprise at least onesuperdistintegrant. Superdisintegrants may, for example, improvedisintegrant efficiency resulting in decreased use levels in comparisonto traditional disintegrants. Examples of superdisintegrants include,but are not limited to, crosscarmelose (a crosslinked cellulose),crospovidone (a crosslinked polymer), sodium starch glycolate (acrosslinked starch), and soy polysaccharides. Commercial examples ofsuperdisintegrants include Kollidon® (BASF), Polyplasdone® XL (ISP), andAc-Di-Sol (FMC BioPolymer).

In some embodiments of the present disclosure, the composition comprisesfrom about 1% to about 25% of at least one superdisintegrant by weightof the composition, such as from about 1% to about 20% by weight, orfrom about 1% to about 15% by weight of the composition. In someembodiments, the compositions comprising at least one superdisintegrantare in a tablet form.

Surfactant/Preconcentrate

The present disclosure further provides for a preconcentratecomposition. In some embodiments of the present disclosure, thecomposition further comprises at least one surfactant to form apreconcentrate. As used herein, the term “preconcentrate” refers to acomposition comprising a fatty acid oil mixture, at least one free fattyacid, and at least one surfactant.

A surfactant may, for example, lower the surface tension of a liquid orthe surface tension between two liquids. For example, surfactantsaccording to the present disclosure may lower the surface tensionbetween the fatty acid oil mixture and/or the at least one free fattyacid and an aqueous solution.

Chemically speaking, surfactants are molecules with at least onehydrophilic part and at least one hydrophobic (i.e., lipophilic) part.Surfactant properties may be reflected in the hydrophilic-lipophilicbalance (HLB) value of the surfactant, wherein the HLB value is ameasure of the degree of hydrophilic versus lipophilic properties of asurfactant. The HLB value normally ranges from 0 to 20, where a HLBvalue of 0 represents high hydrophilic character, and a HLB of 20represents high lipophilic character. Surfactants are often used incombination with other surfactants, wherein the HLB values are additive.The HLB value of surfactant mixtures may be calculated as follows:HLB_(A) (fraction of surfactant A)+HLB_(B) (fraction of surfactantB)=HLB_(A+B) mixture

Surfactants are generally classified as ionic surfactants, e.g., anionicor cationic surfactants, and nonionic surfactants. If the surfactantcontains two oppositely charged groups, the surfactant is named azwitterionic surfactant. Other types of surfactants include, forexample, phospholipids.

In at least one embodiment of the present disclosure, the compositioncomprises at least one surfactant chosen from nonionic, anionic,cationic, and zwitterionic surfactants.

Non-limiting examples of nonionic surfactants suitable for the presentdisclosure are mentioned below.

Pluronic® surfactants are nonionic copolymers composed of a centralhydrophobic polymer (polyoxypropylene(poly(propylene oxide))) with ahydrophilic polymer (polyoxyethylene(poly(ethylene oxide))) on eachside. Various commercially-available Pluronic® products are listed inTable 1.

TABLE 1 Examples of Pluronic ® surfactants. Average Molecular TypeWeight (D) HLB Value Pluronic ® L-31 Non-ionic 1100 1.0-7.0 Pluronic ®L-35 Non-ionic 1900 18.0-23.0 Pluronic ® L-61 Non-ionic 2000 1.0-7.0Pluronic ® L-81 Non-ionic 2800 1.0-7.0 Pluronic ® L-64 Non-ionic 290012.0-18.0 Pluronic ® L-121 Non-ionic 4400 1.0-7.0 Pluronic ® P-123Non-ionic 5800 7-9 Pluronic ® F-68 Non-ionic 8400 >24 Pluronic ® F-108Non-ionic 14600 >24

Brij® are nonionic surfactants comprising polyethylene ethers. Variouscommercially-available Brij® products are listed in Table 2.

TABLE 2 Examples of Brij ® surfactants. HLB Type Compound Value Brij ®30 Non-ionic Polyoxyethylene(4) lauryl ether 9.7 Brij ® 35 Non-ionicpolyoxyethylene (23) lauryl ether 16.9 Brij ® 52 Non-ionicPolyoxyethylene (2) cetyl ether 5.3 Brij ® 56 Non-ionic Polyoxyethylene(10) cetyl ether 12.9 Brij ® 58 Non-ionic Polyoxyethylene (20) cetylether 15.7 Brij ® 72 Non-ionic polyoxyethylene (2) stearyl ether 4.9Brij ® 76 Non-ionic polyoxyethylene (10) stearyl ether 12.4 Brij ® 78Non-ionic polyoxyethylene (20) stearyl ether 15.3 Brij ® 92V Non-ionicPolyoxyethylene (2) oleyl ether 4.9 Brij ® 93 Non-ionic Polyoxyethylene(2) oleyl ether 4 Brij ® 96V Non-ionic polyethylene glycol oleyl ether12.4 Brij ® 97 Non-ionic Polyoxyethylene (10) oleyl ether 12 Brij ® 98Non-ionic Polyoxyethylene (20) oleyl ether 15.3 Brij ® 700 Non-ionicpolyoxyethylene (100) stearyl ether 18

Span® are nonionic surfactants comprising sorbitan esters. Span® isavailable from different sources including Aldrich. Variouscommercially-available Span® products are listed in Table 3.

TABLE 3 Examples of Span ® surfactants. Type Compound HLB Value Span ®20 Non-ionic sorbitan monolaurate 8.6 Span ® 40 Non-ionic sorbitanmonopalmitate 6.7 Span ® 60 Non-ionic sorbitan monostearate 4.7 Span ®65 Non-ionic sorbitan tristearate 2.1 Span ® 80 Non-ionic sorbitanmonooleate 4.3 Span ® 85 Non-ionic Sorbitan trioleate 1.8

Tween® (polysorbates) are nonionic surfactants comprisingpolyoxyethylene sorbitan esters. Various commercially-available Tween®products are listed in Table 4.

TABLE 4 Examples of Tween ® surfactants. Type Compound HLB Value Tween ®20 Non-ionic polyoxyethylene (20) 16.0 sorbitan monolaurate Tween ® 40Non-ionic polyoxyethylene (20) 15.6 sorbitan monopalmitate Tween ® 60Non-ionic polyoxyethylene sorbitan 14.9 monostearate Tween ® 65Non-ionic polyoxyethylene sorbitan 10.5 tristearate Tween ® 80 Non-ionicpolyoxyethylene(20)sorbitan 15.0 monooleate Tween ® 85 Non-ionicpolyoxyethylene sorbane 11.0 trioleate

Myrj® are nonionic surfactants comprising polyoxyethylene fatty acidesters. Various commercially-available Myrj® products are listed inTable 5.

TABLE 5 Examples of Myrj ® surfactants. Type Compound HLB Value Myrj ®45 Non-ionic polyoxyethylene monostearate 11.1 Myrj ® 49 Non-ionicpolyoxyethylene monostearate 15.0 Myrj ® 52 Non-ionic polyoxyethylenemonostearate 16.9 Myrj ® 53 Non-ionic polyoxyethylene monostearate 17.9

Cremophor® are nonionic surfactants. Various commercially-availableCremophor® products are listed in Table 6.

TABLE 6 Examples of Cremophor ® surfactants. Type Compound HLB ValueCremophor ® Non-ionic polyoxyethylated castor oil  2-14 REL Cremophor ®Non-ionic hydrogenated 14-16 RH40 polyoxyethylated castor oilCremophor ® Non-ionic hydrogenated 15-17 RH60 polyoxyethylated castoroil Cremophor ® Non-ionic hydrogenated 16.1 RO polyoxyethylated castoroil

According to the present disclosure, other exemplary nonionicsurfactants include, but are not limited to, diacetyl monoglycerides,diethylene glycol monopalmitostearate, ethylene glycolmonopalmitostearate, glyceryl behenate, glyceryl distearate, glycerylmonolinoleate, glyceryl mono-oleate, glyceryl monostearate, macrogolcetostearyl ether such as cetomacrogol 1000 and polyoxy 20 cetostearylether, macrogol 15 hydroxystearate, macrogol lauril ethers such aslaureth 4 and lauromacrogol 400, macrogol monomethyl ethers, macrogololeyl ethers such as polyoxyl 10 oleyl ether, macrogol stearates such aspolyoxyl 40 stearate, menfegol, mono and diglycerides, nonoxinols suchas nonoxinol-9, nonoxinoll 0 and nonoxinol-11, octoxinols such asoctoxinol 9 and oxtoxinol 10, polyoxamers such as polyoxalene,polyoxamer 188, polyoxamer 407, polyoxyl castor oil such as polyoxyl 35castor oil, polyoxyl hydrogenated castor oil such as polyoxyl 40hydrogenated castor oil, propylene glycol diacetate, propylene glycollaurates such as propylene glycol dilaurate and propylene glycolmonolaurate. Further examples include propylene glycolmonopalmitostearate, quillaia, sorbitan esters, and sucrose esters.

Anionic surfactants suitable for the present disclosure include, forexample, salts of perfluorocarboxylic acids and perfluorosulphonic acid,alkyl sulphate salts such as sodium dodecyl sulphate and ammonium laurylsulphate, sulphate ethers such as sodium lauryl ether sulphate, andalkyl benzene sulphonate salts.

Cationic surfactants suitable for the present disclosure include, forexample, quaternary ammonium compounds such as benzalkonium chloride,cetylpyridinium chlorides, benzethonium chlorides, and cetyltrimethylammonium bromides or other trimethylalkylammonium salts.

Zwitterionic surfactants include, but are limited to, for exampledodecyl betaines, coco amphoglycinates and cocamidopropyl betaines.

In some embodiments of the present disclosure, the surfactant maycomprise a phospholipid, derivative thereof, or analogue thereof. Suchsurfactants may, for example, be chosen from natural, synthetic, andsemisynthetic phospholipids, derivatives thereof, and analogues thereof.Exemplary phospholipids surfactants include phosphatidylcholines withsaturated, unsaturated and/or polyunsaturated lipids such asdioleoylphosphatidylcholine, dipentadecanoylphosphatidylcholine,dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine,dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,di-eicopentaenoyl(EPA)choline, didocosahexaenoyl(DHA)choline,phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserinesand phosphatidylinositols. Other exemplary phospholipid surfactantsinclude soybean lecithin, egg lecithin, diolelyl phosphatidylcholine,distearoyl phosphatidyl glycerol, PEG-ylated phospholipids, anddimyristoyl phosphatidylcholine.

Phospholipids may be “natural” or from a marine origin chosen from, e.g.phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine andphosphatidylinosytol. The fatty acid moiety may be chosen from 14:0,16:0, 16: 1n-7, 18:0, 18:1n-9, 18:1n-7, 18:2n-6, 18:3n-3, 18:4n-3,20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3, or any combinations thereof. Inone embodiment, the fatty acid moiety is chosen from palmitic acid, EPAand DHA.

Other exemplary surfactants suitable for the present disclosure arelisted in Table 7.

TABLE 7 Other surfactants Surfactant Type HBL Value Ethylene glycoldistearate Nonionic 1.5 Glyceryl monostearate Nonionic 3.3 Propyleneglycol monostearate Nonionic 3.4 Glyceryl monostearate Nonionic 3.8Diethylene glycol monolaurate Nonionic 6.1 Acacia Anionic 8.0Cetrimonium bromide Cationic 23.3 Cetylpyridinium chloride Cationic 26.0Polyoxamer 188 Nonionic 29.0 Sodium lauryl sulphate Anionic 40

In some embodiments of the present disclosure, the at least onesurfactant does not comprise Labrasol, Cremophor RH40, or thecombination of Cremophor and Tween-80.

In some embodiments, the at least one surfactant has ahydrophilic-lipophilic balance (HLB) of less than about 10, such as lessthan about 9, or less than about 8.

Co-Surfactant

In some embodiments, compositions of the present disclosure furthercomprise at least one co-surfactant. As used herein the term“co-surfactant” means a substance added to, e.g., the preconcentrate incombination with the at least one surfactant to affect, e.g., increaseor enhance, emulsification and/or stability of the preconcentrate, forexample to aid in forming an emulsion. In some embodiments, the at leastone co-surfactant is hydrophilic.

Examples of co-surfactants suitable for the present disclosure include,but are not limited to, short chain alcohols comprising from 1 to 6carbons (e.g., ethanol), benzyl alcohol, alkane diols and trials (e.g.,propylene glycol, glycerol, polyethylene glycols such as PEG and PEG400), glycol ethers such as tetraglycol and glycofurol (e.g.,tetrahydrofurfuryl PEG ether), pyrrolidine derivatives such as N-methylpyrrolidone (e.g., Pharmasolve®) and 2-pyrrolidone (e.g., Soluphor® P),and bile salts, for example sodium deoxycholate. Further examplesinclude ethyl oleate.

In some embodiments, the at least one co-surfactant comprises from about1% to about 10%, by weight relative to the weight of the preconcentrate.

Solvent

In some embodiments, compositions of the present disclosure, such as thepreconcentrate, further comprises at least one solvent. Hydrophilicsolvents suitable for the present disclosure include, but are notlimited to, alcohols, including water-miscible alcohols, such asabsolute ethanol and/or glycerol, and glycols, for example glycolsobtainable from an oxide such as ethylene oxide, such as 1,2-propyleneglycol. Other non-limiting examples include polyols, such aspolyalkylene glycol, e.g., poly(C₂₋₃)alkylene glycol such aspolyethylene glycol.

In some embodiments of the present disclosure, the preconcentratecomprises at least one substance that acts both as a co-surfactant and asolvent, for example an alcohol such as ethanol. In other embodiments,the preconcentrate comprises at least one co-surfactant and at least onesolvent that are different substances. For example, in some embodimentsthe preconcentrate comprises ethanol as the co-surfactant and glycerolas the solvent.

In some embodiments of the present disclosure, the preconcentrate is apharmaceutical preconcentrate comprising a fatty acid oil mixturecomprising at least 75% eicosapentaenoic acid (EPA) and docosahexaenoicacid (DHA), by weight of the fatty acid oil mixture, wherein the EPA andDHA are in a form chosen from ethyl ester and triglyceride; at least onefree fatty acid; and at least one surfactant.

In one embodiment, the pharmaceutical preconcentrate comprises: a fattyacid oil mixture comprising at least 95% of EPA ethyl ester, DHA ethylester, or mixtures thereof, by weight of the fatty acid oil mixture; atleast one free fatty acid chosen from linoleic, α-linolenic acid (ALA),γ-linoleic acid (GLA), and oleic acid; and a least one surfactant chosenfrom polysorbate 20, polysorbate 80, and mixtures thereof.

In another embodiment, the pharmaceutical preconcentrate comprises: afatty acid oil mixture comprising from about 80% to about 88% EPA andDHA by weight of the fatty acid oil mixture, wherein the EPA and DHA arein ethyl ester form; at least one free fatty acid comprising oleic acid;and at least one surfactant chosen from polysorbate 20, polysorbate 80,and mixtures thereof; wherein the at least one surfactant comprises lessthan 40%, by weight relative to the weight of the preconcentrate.

In another embodiment, the pharmaceutical preconcentrate comprises: afatty acid oil mixture comprising from about 80% to about 88% EPA andDHA by weight of the fatty acid oil mixture, wherein the EPA and DHA arein ethyl ester form; at least one free fatty acid comprising linoleicacid; and at least one surfactant chosen from polysorbate 20,polysorbate 80, and mixtures thereof; wherein the at least onesurfactant comprises less than 35%, by weight relative the weight of thepreconcentrate.

In another embodiment, the pharmaceutical preconcentrate comprises: afatty acid oil mixture comprising from about 80% to about 88% EPA andDHA by weight of the fatty acid oil mixture, wherein the EPA and DHA arein ethyl ester form; at least one free fatty acid comprising from about80% to about 88% EPA and DHA, by weight of the at least one free fattyacid, wherein the EPA and DHA are in free acid form; and at least onesurfactant chosen from polysorbate 20, polysorbate 80, and mixturesthereof. For example, the pharmaceutical preconcentrate may compriseK85EE as the fatty acid oil mixture, K85FA as the at least one freefatty acid, and at least one surfactant chosen from polysorbate 20,polysorbate 80, and mixtures thereof.

In another embodiment, the pharmaceutical preconcentrate may compriseK85EE as the fatty acid oil mixture, K85FA as the at least one freefatty acid, and at least one surfactant chosen from polysorbate 20 orpolysorbate 80, wherein the [K85EE]:[Tween]:[K85FA] ranges from e.g.about 5:2:0.5 to 5:4:2. In a further embodiment, the ration between[K85EE]:[Tween]:[K85FA] is about [4-5]:[3-4]:[1-1.5].

In another embodiment, minimum of about 5-10% up to maximum of about 50%of fatty acid oil mixture comprising from about 80% to about 88% EPA andDHA by weight of the fatty acid oil mixture, wherein the EPA and DHA arein ethyl ester form, is substituted by a free fatty acid chosen from aK85-FA composition (corresponding to a K85-FA fatty acid profileachieved by hydrolyzing a K85-EE fatty acid ethyl ester composition)EPA, DPA, DHA, and combinations thereof. For example, the EPA-EE andDHA-EE content from 400 mg/g to 840 mg/g of total fatty acid oil mixtureis replaced by 40 to 440 mg/g Free fatty acid chosen from a K85-FAcomposition.

In other embodiments, the preconcentrate is a food supplement ornutritional supplement preconcentrate comprising a fatty acid oilmixture comprising from about 25% to about 75% eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA), by weight of the fatty acid oilmixture, wherein the EPA and DHA are in a form chosen from ethyl esterand triglyceride; at least one free fatty acid; and at least onesurfactant.

In some embodiments, the weight ratio of fatty acid oilmixture:surfactant of the preconcentrate ranges from about 1:1 to about10:1, from about 1.1 to about 8:1, from 1:1 to about 7:1, from 1:1 toabout 6:1, from 1:1 to about 5:1, from 1:1 to about 4:1, from 1:1 toabout 3:1, or from 1:1 to about 2:1.

In some embodiments, the at least one surfactant comprises from about 5%to about 55%, by weight relative to the total weight of thepreconcentrate. For example, in some embodiments, the at least onesurfactant comprises from about 5% to about 35%, from about 10% to about35%, from about 15% to about 35%, from about 15% to about 30%, or fromabout 20% to about 30%, by weight, relative to the total weight of thepreconcentrate.

SNEDDS/SMEDDS/SEDDS

The preconcentrate of the present disclosure may be in a form of aself-nanoemulsifying drug delivery system (SNEDDS), aself-microemulsifying drug delivery system (SMEDDS), or a selfemulsifying drug delivery system (SEDDS), wherein the preconcentrateforms an emulsion in an aqueous solution.

Without being bound by theory, it is believed that the preconcentrateforms a SNEDDS, SMEDDS, and/or SEDDS upon contact with gastric and/orintestinal media in the body, wherein the preconcentrate forms anemulsion comprising micelle particles. The emulsion may, for example,provide for increased or improved stability of the fatty acids foruptake in the body and/or provide increased or improved surface area forabsorption. SNEDDS/SMEDDS/SEDDS may thus provide for enhanced orimproved hydrolysis, solubility, bioavailability, absorption, or anycombinations thereof of fatty acids in vivo.

Generally, known SNEDDS/SMEDDS/SEDDS formulations comprise ˜10 mg of adrug and ˜500 mg of surfactants/co-surfactants. The SNEDDS/SMEDDS/SEDDSpresently disclosed may have the opposite relationship, i.e., the amountof fatty acid oil mixture comprising the active pharmaceuticalingredient (API) is greater than the amount of surfactant.

The SNEDDS/SMEDDS/SEDDS presently disclosed may comprise a particle size(i.e., particle diameter) ranging from about 5 nm to about 10μm. Forexample, in some embodiments, the particle size ranges from about 5 nmto about 1μm, such as from about 50 nm to about 750 nm, from about 100nm to about 500 nm, or from about 150 nm to about 350 nm.

Excipients

The compositions, preconcentrates, and/or SNEDDS/SMEDDS/SEDDS presentlydisclosed may further comprise at least one non-active pharmaceuticalingredient, e.g., excipient. Non-active ingredients may solubilize,suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color,flavor, and/or fashion active ingredients into an applicable andefficacious preparation, such that it may be safe, convenient, and/orotherwise acceptable for use. The at least one non-active ingredient maybe chosen from colloidal silicon dioxide, crospovidone, lactosemonohydrate, lecithin, microcrystalline cellulose, polyvinyl alcohol,povidone, sodium lauryl sulfate, sodium stearyl fumarate, talc, titaniumdioxide, and xanthum gum.

The compositions, preconcentrates, and/or SNEDDS/SMEDDS/SEDDS presentlydisclosed may further comprise at least one antioxidant. Examples ofantioxidants suitable for the present disclosure include, but are notlimited to, α-tocopherol (vitamin E), calcium disodium EDTA, alphatocoferyl acetates, butylhydroxytoluenes (BHT), and butylhydroxyanisoles(BHA).

The compositions presently disclosed may be administered, e.g., incapsule, tablet or any other drug delivery forms. For example, thecompositions and/or preconcentrates presently disclosed may beencapsulated, such as a gelatin capsule.

In some embodiments of the present disclosure, the capsule fill contentranges from about 0.400 g to about 1.600 g. For example, in someembodiments, the capsule fill content ranges from about 0.400 g to about1.300 g, from about 0.600 g to about 1.200 g, from about 0.600 g toabout 0.800 g, from about 0.800 g to about 1.000, from about 1.000 g toabout 1.200 g, or any amount in between. For example, in someembodiments the capsule fill content is about 0.600 g, about 0.800 g,about 1.000 g, or about 1.200 g.

The capsules presently disclosed may be manufactured in low oxygenconditions to inhibit oxidation during the manufacturing process.Preparation of capsules and/or microcapsules in accordance with thepresent disclosure may be carried out following any of the methodsdescribed in the literature. Examples of such methods include, but arenot limited to, simple coacervation methods (see, e.g., ES 2009346, EP0052510, and EP 0346879), complex coacervation methods (see, e.g., GB1393805), double emulsion methods (see, e.g., U.S. Pat. No. 4,652,441),simple emulsion methods (see, e.g., U.S. Pat. No. 5,445,832), andsolvent evaporation methods (see, e.g., GB 2209937). Those methods may,for example, provide for continuous processing and flexibility of batchsize.

Methods or Uses

The present disclosure further encompasses methods of treating and/orregulating at least one health problem in a subject in need thereof. Thecompositions presently disclosed may be administered, e.g., in capsule,tablet or any other drug delivery forms, to a subject for therapeutictreatment and/or regulation of at least one health problem including,for example, irregular plasma lipid levels, cardiovascular functions,immune functions, visual functions, insulin action, neuronaldevelopment, heart failure, and post myocardial infarction. In someembodiments, the at least one health problem is chosen from mixeddyslipidemia, dyslipidemia, hypertriglyceridemia, hypercholesterolemia,heart failure, and post-myocardial infarction.

In one embodiment, the present disclosure provides for a method oftreating at least one health problem in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising a pharmaceutically-effective amount of a fatty acid oilmixture comprising at least 75% eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), by weight of the fatty acid oil mixture,wherein the EPA and DHA are in a form chosen from ethyl ester andtriglyceride; and at least one free fatty acid. In some embodiments, themethod treats at least one of elevated triglyceride levels, non-HDLcholesterol levels, LDL cholesterol levels and/or VLDL cholesterollevels. For example, the method may reduce triglyceride levels fromabout 30% to about 80%, such as from about 40% to about 70%, from about40% to about 60%, or from about 30% to about 50%, in a subject withelevated triglyceride levels.

In another embodiment, the present disclosure provides for a method ofregulating at least one health problem in a subject in need thereof,comprising administering to the subject administering to the subject asupplement composition comprising: a fatty acid oil mixture comprisingfrom about 25% to about 75% eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), by weight of the fatty acid oil mixture,wherein the EPA and DHA are in a form chosen from ethyl ester andtriglyceride; and at least one free fatty acid; wherein the at least onehealth problem is chosen from irregular plasma lipid levels,cardiovascular functions, immune functions, visual functions, insulinaction, neuronal development, heart failure, and post myocardialinfarction.

In still a further embodiment, the present disclosure provides for amethod for enhancing at least one parameter chosen from hydrolysis,solubility, bioavailability, absorption, and combinations thereof ofeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) comprisingcombining: a fatty acid oil mixture comprising EPA and DHA in a formchosen from ethyl ester and triglyceride; and at least one free fattyacid. For example, combining: a fatty acid oil mixture comprising EPAand DHA in a form chosen from ethyl ester and triglyceride; at least onefree fatty acid; and at least one surfactant; wherein the fatty acid oilmixture, that at least one free fatty acid, and the at least onesurfactant form a preconcentrate. In addition, the preconcentrate canform a self-nanoemulsifying drug delivery system (SNEDDS),self-microemulsifying drug delivery system (SMEDDS), or self-emulsifyingdrug delivery system (SEDDS) in an aqueous solution. The bioavailablitymay be increased by at least 40%, such as by about 80% or by at least85%.

In some embodiments, the pharmaceutical composition or supplementcomposition further comprises at least one surfactant to form apreconcentrate for administration to a subject in need thereof to treatand/or regulate at least one health problem. In some embodiments, thepreconcentrate forms a self-nanoemulsifying drug delivery system(SNEDDS), a self-microemulsifying drug delivery system (SMEDDS), or aself-emulsifying drug delivery system (SEDDS) in an aqueous solution. Insome embodiments, the aqueous solution is gastric media and/orintestinal media.

The total daily dosage of the fatty acid oil mixture may range fromabout 0.600 g to about 6.000 g. For example, in some embodiments, thetotal dosage of the fatty acid oil mixture ranges from about 0.800 g toabout 4.000 g, from about 1.000 g to about 4.000 g, or from about 1.000g to about 2.000 g. In one embodiment, the fatty acid oil mixture ischosen from K85EE and AGP 103 fatty acid oil compositions.

The composition and/or preconcentrates presently disclosed may beadministered in from 1 to 10 dosages, such as from 1 to 4 times a day,such as once, twice, three times, or four times per day, and further forexample, once, twice or three times per day. The administration may beoral or any other form of administration that provides a dosage of fattyacids, e.g., omega-3 fatty acids, to a subject.

The following examples are intended to illustrate the present disclosurewithout, however, being limiting in nature. It is understood that theskilled artisan will envision additional embodiments consistent with thedisclosure provided herein.

EXAMPLES Example 1: Preconcentrates

Different preconcentrates were prepared as described in Table 9. Toprepare the preconcentrates, the components were mixed according to theschemes identified below on a weight to weight basis. Thepreconcentrates were visually inspected after mixing and again afterbeing stored for 24 hours at room temperature. Under the Preconcentrateheading, a “clear” designation represents a transparent homogenousmixture; an “unclear” designation represents a nonhomogenous mixture,where some turbidity can be observed by visual inspection. The degree ofturbidity was not determined.

All clear preconcentrates were emulsified in gastric media, by addinggastric media (2 ml) to approximately 100 mg of the preconcentrate. Thecomposition of the gastric media is shown in Table 8.

TABLE 8 Composition of Gastric Media. Gastric Media Bile salts, Porcine(mM) 0.08 Lechitin (mM) 0.02 Sodium chloride (mM) 34.2 Pepsin (mg/ml)0.1 pH 1.6 (adjust with 1M HCl) Osmolarity (mOsm/kg) 120

The outcome of the emulsification was recorded approximately 3 hoursafter mixing. A majority of the preconcentrates formed milky emulsionsimmediately after mixing. Emulsions that stayed milky and homogenousafter 3 hours are described as “milky,” under the Emulsion heading.Emulsions that separated or became nonhomogenous or where oil drops wereobserved are described as “separates,” under the Emulsion heading.

Selected emulsions were further characterized by determining theparticle size. Particle size was measured using a Malvern Zetasizer(Malvern Instrument, Worcestershire, UK) with particle size measuringrange of 0.5-6000 nm and Zeta potential of particle range of 3 nm-10 μm.The particle size was measured in triplicate. The K85EE (EE=ethyl ester)fatty acid composition used herein is sold in a gelatin capsule andbranded primarily under the trademarks Lovaza™ or Omacor®.

TABLE 9 Preconcentrates. K85- Tween- Total Particle EE 20 Oleic Acidvol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm) 1 451.4234.3 99 784.7 57:29:12 Unclear — — 2 448.8 299.7 53.8 802.3 55:37:6Unclear — — 3 451.2 324.7 24.7 800.6 56:40:3 Unclear — — 10 400 300 100800 50:37:12 Clear Milky 271 11 404 298 97 799 50:37:12 Clear Milky — 12500 300 217 1017 49:29:21 Clear Separates — 13 398 300 99 797 49:37:12Clear Milky 257 14 399 252 98 749 53:33:13 Clear Separates 226 15 400204 102 706 56:28:14 Clear Separates 199 21 450 198 133 781 57:25:17Clear Separates — 23 549 204 169 922 59:22:18 Clear Separates — 24 600200 178 978 61:20:18 Clear Separates — 26 453 214 121 788 57:27:15 ClearSeparates — 27 456 220 121 797 57:27:15 Clear Separates — 28 452 228 144824 54:27:17 Clear Separates — 29 448 230 122 800 56:28:15 ClearSeparates — 30 452 242 124 818 55:29:15 Clear Separates — 31 449 251 124824 54:30:15 Clear Milky — 32 448 260 123 831 53:31:14 Clear Separates —33 452 270 121 843 53:32:14 Clear Separates — 34 449 281 123 85352:32:14 Clear Separates — 35 448 290 121 859 52:33:14 Clear Separates —K85- Total Particle EE Tween- Ricinoleic Vol. Pre- Size No. (mg) 20 (mg)Acid (mg) (mg) Ratio conc. Emulsion (nm) 36 402 298 98 798 50:37:12Clear Milky 277 37 402 250 100 752 53:33:13 Clear Milky 268 38 400 200100 700 57:28:14 Unclear — — 39 450 250 100 800 56:31:12 Clear Milky —43 400 110 100 610 65:18:16 Clear Separates — 44 500 270 105 87557:30:12 Clear Separates — 45 505 295 103 903 55:32:11 Clear Milky — 46525 250 143 918 57:27:15 Clear Separates — 47 500 252 118 870 57:28:13Clear Separates — 48 297 293 145 735 40:39:19 Clear Separates — 49 500260 127 887 56:29:14 Clear Separates — 50 499 285 106 890 56:32:11 ClearSeparates — 51 403 298 193 894 45:33:21 Clear Milky — 52 460 250 90 80057:31:11 Clear — — K85- Tween- Ricinoleic Total Particle EE 40 acid Vol.Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm) 53 450 25598 803 56:31:12 Clear Milky 237 55 498 220 98 816 61:26:12 Clear Milky226 56 505 202 106 813 62:24:13 Clear Separates — 57 500 200 100 80062:25:12 Clear Separates — 58 552 152 102 806 68:18:12 Clear Separates —K85- Tween- Ricinoleic Total Particle EE 60 Acid Vol. Pre- Size No. (mg)(mg) (mg) (mg) Ratio conc. Emulsion (nm) 70 500 200 100 800 62:25:12Clear Milky — 71 500 150 100 750 66:20:13 Clear Separates — 72 529 180104 813 65:22:12 Clear Separates — 73 518 200 102 820 63:24:12 ClearSeparates — K85- Tween- Ricinoleic Total Particle EE 80 Acid Vol. Pre-Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm) 54 450 270 105825 54:32:12 Clear Separates — K85- Cremophor Ricinoleic Total ParticleEE EL Acid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion(nm) 40 399.9 300 106.4 806.3 49:37:13 Unclear — — 41 400 256.9 137793.9 50:32:17 Unclear — — K85- Ricinoleic Total Particle EE SoritolAcid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm) 42400 211 104 715 55:29:14 Clear/solid — — when cooled K85- PEG-Ricinoleic Total Particle EE 400 Acid Vol. Pre- Size No. (mg) (mg) (mg)(mg) Ratio conc. Emulsion (nm) 16 399.9 310.2 162.6 872.7 45:35:18 ClearSeparates — 17 398.3 256.8 157.9 813 48:31:19 Clear Separates — 18 402.4198.7 147.5 748.6 53:26:19 Clear Separates — K85- Tween- Total ParticleEE 20 PEG-400 Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc.Emulsion (nm) 19 398.2 297.9 214.7 910.8 43:32:23 Unclear — — 20 403248.2 145.3 796.5 50:31:18 Unclear — — K85- α-Linoleic Total Particle EETween- Acid Vol. Pre- Size No. (mg) 20 (mg) (mg) (mg) Ratio conc.Emulsion (nm) 74 402 300 100 802 50:37:12 Clear Milky — 75 454 249  98801 56:31:12 Slightly Separates — dense 76 502 204 103 809 62:25:12Slightly Separates — dense K85- Tween- α-Linoleic Total Particle EE 40Acid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm) 77403 299 108 810 49:36:13 Clear/ Separates — Precipitate 78 456 252 110818 55:30:13 Clear/ Separates — Precipitate 79 503 217 103 823 61:26:12Clear/ Separates — Precipitate K85- Tween- α-Linoleic Total Particle EE60 Acid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm)80 402 313 104 819 49:38:12 Clear Separates — 81 459 205 100 76460:26:13 Clear Separates — 82 498 198 106 802 62:24:13 Clear Separates —K85- Tween- α-Linoleic Total Particle EE 80 Acid Vol. Pre- Size No. (mg)(mg) (mg) (mg) Ratio conc. Emulsion (nm) 83 407 317 102 826 49:38:12Clear Milky 261.3 84 455 256 110 821 55:31:13 Clear Milky 260.8 85 498208 102 808 61:25:12 Clear Milky 274.5 K85- Tween- Erucuc Total ParticleEE 20 Acid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion(nm) 86 401 300  99 800 50:37:12 Clear Semi — Milky 87 451 250 105 80655:31:13 Clear Separates — 88 504 204 102 810 62:25:12 Clear Separates —K85- Tween- Erucuc Total Particle EE 40 Acid Vol. Pre- Size No. (mg)(mg) (mg) (mg) Ratio conc. Emulsion (nm) 89 401 298 102 801 50:37:12Clear Separates — 90 451 254  99 804 56:31:12 Clear Separates — 91 504219 103 826 61:26:12 Clear Separates — K85- Tween- Erucuc Total ParticleEE 60 Acid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion(nm) 92 401 301 104 806 49:37:12 Clear Separates — 93 454 267 101 82255:32:12 Clear Separates — 94 497 202 100 799 62:25:12 Clear Separates —K85- Tween- Erucuc Total Particle EE 60 Acid Vol. Pre- Size No. (mg)(mg) (mg) (mg) Ratio conc. Emulsion (nm) 95 406 298 100 804 50:37:12Clear Separates — 96 450 251 102 803 56:31:12 Clear Separates — 97 502205 122 829 60:24:14 Clear Separates — α- K85- Tween- Linolenic TotalParticle EE 20 acid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc.Emulsion (nm) 98 401 308 105 814 49:37:12 Clear Milky, — beginningseparation 102 450 264 108 822 54:32:13 Clear Milky, — beginningseparation 106 501 200 111 812 61:24:13 Clear Milky, with — separationα- K85- Tween- Linolenic Total Particle EE 40 Acid Vol. Pre- Size No.(mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm) 99 402 302 102 80649:37:12 Clear Milky, — beginning separation 103 452 254 101 80756:31:12 Clear Milky, with — separation 107 502 206 108 816 61:25:13Clear Milky, with — separation α- K85- Tween- Linolenic Total ParticleEE 60 Acid Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion(nm) 100 403 303 103 809 49:37:12 Clear Milky, — beginning separation104 450 249 102 801 56:31:12 Clear Milky, with — separation 108 506 200100 806 62:24:12 Unclear Milky, — beginning separation α- K85- Tween-Linolenic Total Particle EE 80 Acid Vol. Pre- Size No. (mg) (mg) (mg)(mg) Ratio conc. Emulsion (nm) 101 403 308 106 817 49:37:12 Clear Milky,— beginning separation 105 452 253 102 807 56:31:12 Clear Milky, with —separation 109 507 203 112 822 61:24:13 Clear Milky, with — separationK85- Tween- Total Particle EE 20 KE85-FA Vol. Pre- Size No. (mg) (mg)(mg) (mg) Ratio conc. Emulsion (nm) 110 398.5 300.5  98.6 797.6 49:37:12Clear Milky (<10 min waiting time) 111 448 245.9 110.4 804.3 55:30:13Unclear — — 112 498.3 197.9 106.2 802.4 62:24:13 Unclear — — K85- Tween-Total Particle EE 40 KE85-FA Vol. Pre- Size No. (mg) (mg) (mg) (mg)Ratio conc. Emulsion (nm) 113 405.7 303.7 105.8 815.2 49:37:12 ClearMilky — (<10 min waiting time) 114 452.8 261.6 101.8 816.2 55:32:12Clear Milky — (<10 min waiting time) 115 499 212.2 114.7 825.9 60:25:13Clear Milky — (<10 min waiting time) K85- Tween- Total Particle EE 60KE85-FA Vol. Pre- Size No. (mg) (mg) (mg) (mg) Ratio conc. Emulsion (nm)116 395 296.2 100 791.2 49:37:12 Clear Milky — (<10 min waiting time)117 450.3 253.1 98.2 801.6 56:31:12 Clear Milky — (<10 min waiting time)118 500.8 206 105.7 812.5 61:25:13 Clear Milky — (<10 min waiting time)K85- Tween- Total Particle EE 80 KE85-FA Vol. Pre- Size No. (mg) (mg)(mg) (mg) Ratio conc. Emulsion (nm) 119 402 308.3 100.8 811.1 49:38:12Clear Milky, — sticky (<10 min waiting time) 120 456.6 260.3 103.5 820.455:31:12 Clear Milky, — sticky (<10 min waiting time) 121 502.3 202.2104 808.5 62:25:12 Clear Milky, — sticky (<10 min waiting time)

Of the preconcentrates prepared, formulation number 85 facilitated aload of 60% K85EE into the preconcentrate and gave a stable emulsion ingastric media with a particle size determined to be about 275 nm.Attempts to prepare preconcentrates with saturated fatty acids, stearicacid and decanoic acid failed. Although homogenous preconcentrates couldbe obtained by heating, a precipitation of stearic acid or decanoic acidwas observed upon cooling of the preconcentrate to room temperature.

Example 2: Additional Preconcentrates

Additional preconcentrates were prepared to determine an optimizedamount of surfactant with K85EE and K85FA. The preconcentrates describedin Table 10 were prepared as provided in Example 1. The preconcentrateswere visually inspected after mixing and again after being stored for 24hours at room temperature. Under the Preconcentrate heading, a “clear”designation represents a transparent homogenous mixture; a “turbid”designation represents a nonhomogenous mixture, where some turbidity canbe observed by visual inspection. The degree of turbidity was notdetermined.

TABLE 10 Additional Preconcentrates. K85-EE Tween20 K85FA Precon- (mg)(mg) (mg) centrate 107 307 62 Turbid 107 307 76 Turbid 107 307 102Turbid 107 307 200 Clear 107 307 401 Clear 107 307 803 Clear 107 3071608 Clear 26 300 99 Clear 104 300 99 Clear 201 300 99 Clear 316 300 99Clear 400 300 99 Clear 497 300 99 Turbid 618 300 99 Turbid 405 42 101Clear 405 99 101 Clear 405 202 101 Clear 405 299 101 Clear 405 400 101Clear 405 618 101 Clear 405 1000 101 Clear K85-EE Tween80 K85FA Precon-(mg) (mg) (mg) centrate 407 306 57 Clear 407 306 80 Clear 407 306 103Clear 407 306 202 Clear 407 306 401 Clear 28 299 101 Clear 57 299 101Clear 99 299 101 Clear 233 299 101 Clear 316 299 101 Clear 414 299 101Clear 510 299 101 Clear 569 299 101 Clear 627 299 101 Clear 688 299 101Clear 769 299 101 Clear 402 32 106 Clear 402 126 106 Clear 402 229 106Clear 402 326 106 Clear 402 410 106 Clear 402 997 106 Clear K85-EETween-40 K85FA Precon- (mg) (mg) (mg) centrate 111 311 59 Turbid 111 31170 Clear 111 311 95 Clear 111 311 135 Clear 111 311 244 Clear 111 311798 Clear 111 311 1567 Clear 30 309 98 Clear 110 309 98 Clear 208 309 98Clear 322 309 98 Clear 404 309 98 Clear 501 309 98 Turbid 618 309 98Turbid 408 38 99 Clear 408 105 99 Clear 408 210 99 Clear 408 301 99Clear 408 398 99 Clear 408 616 99 Clear 408 1001 99 Clear

Example 3: Compatibility of Preconcentrates with Solvents

The compatibility of solvents and a preconcentrate having a fixed amountof K85EE and Tween-80 were evaluated. The preconcentrates described inTable 11 were prepared as provided in Example 1, but with the additionof the solvent identified below. The preconcentrates were visuallyinspected after mixing and again after being stored for 24 hours at roomtemperature. Under the Preconcentrate heading, a “clear” designationrepresents a transparent homogenous mixture; a “turbid” designationrepresents a non-homogenous mixture, where some turbidity can beobserved by visual inspection. The degree of turbidity was notdetermined.

TABLE 11 Compatibility of Solvent and Preconcentrates. K85-EE Tween-8096% ethanol 96% ethanol Precon- (mg) (mg) (mg) (%) centrate 400 110 10.72.1 Turbid 400 110 18.7 3.5 Turbid 400 110 28.4 5.3 Turbid 400 110 32.15.9 Turbid 400 110 45.7 8.2 Turbid 400 110 53.5 9.5 Turbid 400 110 61.510.8 Turbid 400 110 69.8 12.0 Turbid 400 110 79.9 13.5 Turbid 400 11091.3 15.2 Turbid 400 110 102.5 16.7 Turbid K85-EE Tween-80 Propyleneglycol Propylene glycol Precon- (mg) (mg) (mg) (%) centrate 400 110 11.12.1 Turbid 400 110 16.7 3.2 T urbid 400 110 23.1 4.3 Turbid 400 110 32.96.1 Turbid 400 110 41.5 7.5 Turbid 400 110 48.6 8.7 T urbid 400 110 59.910.5 Turbid 400 110 72.9 12.5 Turbid 400 110 81.5 13.8 Turbid 400 11093.5 15.5 Turbid 400 110 104.6 17.0 Turbid K85-EE Tween-80 PEG 300 PEG300 Precon- (mg) (mg) (mg) (%) centrate 400 110 13.9 2.7 Turbid 400 11023.7 4.4 Turbid 400 110 35.6 6.5 Turbid 400 110 47.1 8.5 Turbid 400 11055.0 9.7 Turbid 400 110 68.7 11.9 Turbid 400 110 81.8 13.8 Turbid 400110 90.3 15.0 Turbid 400 110 104.0 16.9 Turbid K85-EE Tween-80 Benzylalcohol Benzyl alcohol Precon- (mg) (mg) (mg) (%) centrate 400 110 0 0Clear 400 110 11.4 2.2 Turbid 400 110 18.1 3.4 Turbid 400 110 30.9 5.7Clear 400 110 45.5 8.2 Clear 400 110 55.6 9.8 Clear 400 110 66.7 11.6Clear 400 110 77.4 13.2 Clear 400 110 92.1 15.3 Clear 400 110 99.0 16.3Clear K85-EE Tween-80 Triacetin Triacetin Precon- (mg) (mg) (mg) (%)centrate 400 110 12.3 2.4 Turbid 400 110 24.3 4.5 Turbid 400 110 35.86.6 Turbid 400 110 45.3 8.2 Turbid 400 110 57.0 10.1 Turbid 400 110 68.111.8 Turbid 400 110 80.9 13.7 Turbid 400 110 90.0 15.0 Turbid 400 110101.7 16.6 Turbid K85-EE Tween-80 1-octadecanol 99% 1-octadecanol 99%Precon- (mg) (mg) (mg) (%) centrate 400 110 8.6 1.7 Precip- itate K85-EETween-80 oleyl alcohol 85% oleyl alcohol 85% Precon- (mg) (mg) (mg) (%)centrate 400 100 13.0 2.5 Turbid 400 100 26.5 4.9 Turbid 400 100 37.36.8 Turbid 400 100 49.5 8.8 Turbid 400 100 62.6 10.9 Turbid 400 100 77.713.2 Turbid 400 100 92.2 15.3 Turbid 400 100 105.7 17.2 Turbid K85-EETween-80 1-tetradecanol 97% 1 tetradecanol 97% Precon- (mg) (mg) (mg)(%) centrate 400 100 1.7 0.3 Turbid 400 100 10.3 2.0 Turbid 400 100 22.74.3 Turbid 400 100 35.8 6.6 Precip- itate K85-EE Tween-80 glycerolglycerol Precon- (mg) (mg) (mg) (%) centrate 400 100 17.7 3.4 Turbid 400100 28.0 5.2 Turbid 400 100 41.7 7.6 Turbid 400 100 52.8 9.4 Turbid 400100 71.2 12.3 Turbid 400 100 85.4 14.3 Turbid 400 100 92.3 15.3 Turbid400 100 105.7 17.2 Turbid K85-EE Tween-80 Oleic acid 90% Oleic acid 90%Precon- (mg) (mg) (mg) (%) centrate 400 100 13.2 2.5 Turbid 400 100 23.94.5 Turbid 400 100 31.5 5.8 Turbid 400 100 41.4 7.5 Turbid 400 100 51.89.2 Turbid 400 100 65.2 11.3 Clear 400 100 79.8 13.5 Clear 400 100 87.214.6 Clear 400 100 102.2 16.7 Clear K85-EE Tween-80 1-docosanol 98%1-docosanol 98% Precon- (mg) (mg) (mg) (%) centrate 400 100 9.6 1.8Precip- itate

Example 4: Characterization of Preconcentrates and SNEDDS/SMEDDS/SEDDS

Preconcentrates A-L described in Table 12 were prepared as provided inExample 1.

TABLE 12 Preconcentrates A-L. Precon- K85-EE Surfactant FFA Total vol.centrate (mg) (mg) (mg) (mg) Ratio A 5002.7 Tween-20 Oleic Acid 10016.449:36:13 3705.8 1307.9 B 5004.9 Tween-80 Oleic Acid 10015.1 49:37:133707.9 1302.3 C 5003.2 Tween-20 Ricioleic acid 10013.4 49:36:13 3702.11308.1 D 5003.5 Tween-80 Ricioleic acid 10010 49:36:13 3703.1 1303.4 E5000.4 Tween-20 Linoleic acid 10013.1 49:37:13 3707.4 1305.3 F 5001Tween-80 Linoleic acid 10011.3 49:37:13 3706 1304.3 G 5006.4 Tween-20Erucic acid 10008.7 50:36:12 3702.1 1300.2 H 5004.3 Tween-80 Erucic acid10011.6 49:36:13 3704.1 1303.2 I 5002.9 Tween-20 α-Linolenic acid10013.1 49:36:13 3700.8 1309.4 J 5003.6 Tween-80 α-Linolenic acid10017.3 49:36:13 3701.6 1312.1 K 5002.9 Tween-20 “Pure” EPA- 10013.149:36:13 3700.8 FA + DHA-FA in a ratio close to K85-EE 1309.4 L 5002.9Tween-80 “Pure” EPA- 10013.1 49:36:13 3700.8 FA + DHA-FA in a ratioclose to K85-EE 1309.4

From Table 12 above, all preconcentrates appeared clear and homogenous,except for the formulation with erucic acid. As such, thepreconcentrates can be mixed in any proportion and these mixtures willstill form homogenous and clear preconcentrates.

Preconcentrates A-L were also screened for compatibility with varioussolvents. The outcome of this screening is show in Table 13 below. To500 mg of preconcentrate, approximately 50 mg of each solvent was added.Preconcentrate A was used for all the solvents. Ethanol was tested inall the preconcentrates. The preconcentrates were visually inspectedafter mixing and again after being stored for 24 hours at roomtemperature. Under the Preconcentrate heading, a “clear” designationrepresents a transparent homogenous mixture; an “unclear” designationrepresents a nonhomogenous mixture, where some turbidity can be observedby visual inspection. The degree of turbidity was not determined.

TABLE 13 Preconcentrate Compatibility. Solvent Preconcentrate APreconcentrate B-L 96% Ethanol Clear Clear Benzyl alcohol Clear NdPropylene glycol Unclear Nd Triacetin Clear Nd PEG 300 Unclear NdGlycerol Unclear Nd 1-octadecanol 99% Clear, but solid Nd 1-docosanol98% Unclear Nd Oleyl alcohol 85% Clear Nd 1-tetradecanol 97% Clear NdNd—Not determined.

Viscosity can be used as a physical characterization parameter.Viscosity measurements were taken for preconcentrates A-L in triplicate.Generally, the viscosity showed greater sensitivity for the type offatty acid than for the type of surfactant. FIG. 1 graphicallyillustrates the viscosity of preconcentrates A-L. Although the viscositymeasurements cannot distinguish between Tween 20 versus Tween 80, theviscosity can be impacted by the free fatty acid.

Preconcentrates A-F, I and J were diluted in gastric and intestinalmedia to form an emulsion (i.e., SNEDDS/SMEDDS/SEDDS). The compositionof the gastric media is shown in Table 14, and the composition of theintestinal media is shown in Table 15.

TABLE 14 Gastric Media Gastric Media Bile salts, Porcine (mM) 0.08Lechitin (mM) 0.02 Sodium chloride (mM) 34.2 Pepsin (mg/ml) 0.1 pH 1.6(adjust with 1M HCl) Osmolarity (mOsm/kg) 120

TABLE 15 Intestinal Media Intestinal Media Bile salts, Porcine Bileextract, Sigma 037K0196 (mM) 5 Phospholipids, LIPOID S PC from LIPOID AG(mM) 1.25 Trizma maleate, Sigma Aldrich, T 3128 (mM) 2 Na⁺ (mM) 150

Particle size was measured using a Malvern Zetasizer (MalvernInstrument, Worcestershire, UK) with particle size measuring range of0.5-6000 nm and Zeta potential of particle range of 3 nm-10 μm. Theparticle size was measured in triplicate.

For the gastric media, the emulsions were prepared by adding 1 ml ofgastric media to 50 mg of the preconcentrate. Table 16 below providesthe particle size measurements for preconcentrates A-F, I and J in thegastric media. The particle size measurements in gastric media are alsographically illustrated in FIG. 2.

TABLE 16 Particle size measurements for preconcentrates A-F, I and J ingastric media. Preconcentrates A B C D E F I J Size (nm) 269.6 152.1216.8 271 271.1 287.1 165 244.3 Standard 29.63 5.141 26.24 15.94 6.20836.71 15.87 13.67 Deviation

For the intestinal media, the emulsions were prepared by adding thegastric media (100 μl) obtained above to intestinal media (900 μl).Table 17 below provides the particle size measurements forpreconcentrates A-F, I and J in the intestinal media. The particle sizemeasurements in intestinal media are also graphically illustrated inFIG. 2.

TABLE 17 Particle size measurements for preconcentrates A-F, I and J inintestinal media. Preconcentrates A B C D E F I J Size (nm) 245.9 2314266.7 332.5 233.9 1891 224.3 1788 Standard 7.465 2438 35.38 26.63 10.481936 13.56 930.5 Deviation

As shown in FIG. 2, intestinal media has a larger impact on the particlesize distribution and particularly, preconcentrates comprising Tween 80.That observation has been visualized in FIGS. 3-18. FIGS. 3-18 show theread out from the Malvern zetasizer for four consecutive measurements onthe same sample of each respective preconcentrate. All thepreconcentrates give near to unimodal particle size distributions ingastric media, whereas only preconcentrates comprising Tween 20 staysunimodal when transferred to intestinal media.

Example 5: Lipolysis and Solubilization

Studies were done to analyze the rate of lipolysis (i.e., hydrolysis)and solubilization for different preconcentrates comprising K85EE anddifferent free fatty acids and surfactants. Specifically, fourexperiments were designed to determine how the amount of surfactantinfluences the rate and extent of lipolysis and solubilization. Thelipolysis was conducted on SMEDDS formulations comprising K85EE.

Materials

-   -   Bile salts: Porcine Bile extract (Sigma); contains glycine and        taurine conjugates of hyodeoxycholic acid and other bile salts.    -   Pancreatic lipase, Porcine pancreas (Sigma); contains many        enzymes, including amylase, trypsin, lipase, ribonuclease and        protease.    -   Lechitin: Phospholipids (LIPOID S PC from LIPOID AG)    -   Trizma maleate (Sigma Aldrich)    -   Tween 20, Molecular Biology Grade (AppliChem Darmstadt), Tween        80 (Fluka)    -   α-Linoleic acid (Sigma 60%), Oleic acid (Aldrich 90%)    -   K85-EE and K85-FA

Preconcentrates A-E were prepared as summarized in Table 18.

TABLE 18 Preconcentrates A-E. Precon- Fatty acid oil centrate mixtureFree fatty acid Surfactant A K85EE (400 mg) oleic acid (100 mg) Tween 20(300 mg) B K85EE (400 mg) oleic acid (100 mg) Tween 20 (75 mg) C K85EE(500 mg) linoleic acid (100 mg) Tween 80 (200 mg) D K85EE (400 mg) K85FA(100 mg) Tween 20 (300 mg) E K85EE (400 mg) — Tween 80 (100 mg)

Lipolysis General Procedure

The in vitro dynamic lipolysis model developed by Zangenberg et al.(Zangenberg, N. H. et al., Eur. J. Pharm. Sci. 14, 237-244, 2001;Zangenberg, N. H., et al., Eur. J. Pharm. Sci. 14, 115-122, 2001) wasused with slight modifications. The lipolysis was conducted in athermostated 600 ml jacketed glass vessel in the presence of porcinebile extract, with continuous addition calcium chloride. The lipasesource was porcine pancreatin and the hydrolysis was followed bytitration with sodium hydroxide solution (1.0 N) using a pH stat (pH6.5). The initial composition of the lipolysis media is shown in Table19.

TABLE 19 Initial composition of lipolysis media. Substance InitialConcentration Pancreatic lipase, Porcine pancreas 800 USP units/ml Bilesalts, Porcine Bile extract 5 mM Phospholipids, LIPOID S PC from LIPOIDAG 1.25 mM Trizma maleate 2 mM Na⁺ 150 mM K85-EE 5.58 mg/ml

The final volume in all experiments was 300 ml and the calciumdispensing rate during the experiments was 0.045 mmol/min (0.09 ml/min).In all experiments, the amount of K85-EE added corresponds to 5.58mg/ml.

To determine the course of K85-EE lipolysis by HPLC, crude samples werewithdrawn and acidified with dilute hydrochloric acid. Theconcentrations of EPA-EE, DHA-EE, EPA-FA and DHA-FA were determined byHPLC in triplicate. Experiments were performed with LC AgilentTechnologies 1200 series at a column temperature of 30° C., mobile phase(A) water (0.1% acetic acid) and (B) MeCN (0.1% acetic acid), withgradient: 0 to 8 minutes, from 70% B to 100% B; 8 to 15 minutes, 100% B;16 to 16 minutes: from 100% B to 70% B, 16 to 20 minutes: 70% B. Theflow rate was 0.5 ml/min, UV@ 210 nM, injection volume: 5 μl, and runtime: 20 minutes.

Concentrations of EPA ethyl ester (EPA-EE), DHA ethyl ester (DHA-EE),EPA free acid (EPA-FA), and DHA free acid (DHA-FA) were monitored overtime and the rate of lipolysis calculated as shown in Table 20 forcomparison with Omacor®.

TABLE 20 Lipolysis of EPA and DHA ethyl ester in comparison to Omacor ®.EPA-EE lipolysis DHA-EE lipolysis % lipolysis K85EE (μg/ml/min)(μg/ml/min) at t = 233 min Omacor ® 1.5 2.3 17 A 2.8 4.5 41 B 2.9 3.9 35C 3.7 5.0 47 D 3.5 5.0 55 E 3.8 4.3 45

FIGS. 19, 22, 25, 28, 31, and 34 graphically illustrate thedisappearance of EPA-EE and DHA-EE and the appearance of EPA-FA andDHA-FA during lipolysis of each respective sample examined. Samplepoints from 2 minutes to 233 minutes were included in the graphs. Inaddition, linear regression lines have been included.

FIGS. 20, 23, 26, 29, 32, and 35 provide the percent recovery of EPA+DHAat different time-points for each respective sample examined. Data aregiven as the sum of EPA-EE, DHA-EE, EPA-FA, and DHA-FA and given as apercentage of theoretical amount 5580 pg/ml.

FIGS. 21, 24, 27, 30, 33, and 36 graphically illustrate the percentlipolysis at different time points for EPA-EE, DHA-EE and total K85EE.Values are calculated relative to the total amount of EPA-EE and DHA-EEdetermined by HPLC after lipolysis for 2 minutes.

Example 6: Fatty Acid Oil Mixtures of PharmaceuticalCompositions/Preconcentrates

Fatty acid oil mixtures of pharmaceutical compositions orpreconcentrates, wherein the fatty acid oil mixture is a K85-EEcomposition are presented in Table 21.

TABLE 21 Fatty acid oil mixture for pharmaceuticalcompositions/preconcentrates Fatty acid oil mixture: Minimum Maximum1000 mg K85EE fatty acid oil mixture Value Value EPAEE + DHAEE 800 mg/g880 mg/g EPA EE 430 mg/g 495 mg/g DHA EE 347 mg/g 403 mg/g Total omega-3EE >90% (w/w) EE = ethyl ester

Example 7: Tablet Formulations

Tablets were prepared by immersing the tablet shown in Table 22 in K85EEoil. The mean liquid loading was 160 mg oil/tablet, corresponding toabout 72 v/v %. The tablet can also be prepared without asuperdisintegrant.

TABLE 22 Tablet compositions Tablet composition Example Neusilin US 89%Ac-Di-Sol (croscarmellose sodium) = superdisintegrants 10% Mg-stearate1.0% 

Example 8: Novel K85 Tablet Formulation

A tablet formulation is prepared with the components identified in Table23 by immersing a tablet in a K85EE or AGP oil and an oil in free acidform.

TABLE 23 K83 tablet formulation K85 or AGP oil loading per tabletMinimum Maximum value EPA EE and DHA EE 125 mg 600 mg Free fatty acidoil 2% corresponding 15% corresponding to about 2.5 mg to about 90 mg

Example 9: Preparation of SEDDS and SMEDDS

The preconcentrate can be prepared by mixing a fatty acid oil mixturetogether with at least one surfactant and a free fatty acid.

The preconcentrate can be visually inspected after mixing and againafter being stored at 24 hours at room temperature and clear andtransparent preconcentrate can be obtained.

To the preconcentrate can then an aqueous medium be added to form anoil-in-water emulsion. The dispersion rate for the formation of theoil-in-water emulsion can be very fast, less than one minute.

The microemulsions formed can then be tested regarding hydrolysis, alsocalled lipolysis.

For example, to determine the course of KE85-EE hydrolysis by HPLC,crude samples can be withdrawn and acidified with dilute hydrochloricacid. The concentrations of EPA- ethyl ester, DHA ethyl ester, EPA-freefatty acid and DHA-free fatty acid can then determined by HPLC.

All samples withdrawn from a non-homogenous phase and some variabilityin recovery can be expected, especially at early time points.

TABLE 24 Initial concentrations of components in the hydrolysis medium.Substance Initial concentration Pancreatic lipase, Porcine pancreas,Sigma 800 USP units/ml 095K1149 Bile salts, Porcine Bile extract, Sigma037K0196 5 mM Phospholipids, LIPOID S PC from LIPOID AG 1.25 mM Trizmamaleate, Sigma Aldrich, T 3128 2 mM Na⁺ 150 mM KE85-EE 5.58 mg/ml

An example HPLC analytical method can include the following parameters:

Use of a LC-MS manufactured by Agilent Technologies and includes a 1200Series LC and a 6140 Quadropole MS running ChemStation B.04.01 software;

Column: EclipseXDB C18, 2.1×150 mm, 5 μm, Agilent

Column temperature: 25° C.;

Mobile Phase: A: water (0.1% acetic acid), B: MeCN (0.1% acetic acid);

Gradient: 0 to 8 min, from 70% B to 100% B, 8 to 15 minutes: 100% B,from 16 to 16 minutes: from 100% B to 70% B, 16 to 20 minutes: 70% B;

Flow rate: 0.5 ml/min;

UV@ 210 nM;

Injection volume: 25 μl; and

Run time: 20 minutes.

The oil-in-water emulsions can then be further analyzed to determine theparticle size of the oil droplets. The particle size can be determinedwith Malvern Zetasizer (Malvern Instrument, Worcestershire, UK) havingparticle size measuring range of 0.6-6000 nm and Zeta of particle rangeof 3nm-10 μm.

Table 25 shows the components that can be included in pharmaceuticalcompositions and food supplement compositions according to the presentdisclosure.

TABLE 25 Sample compositions according to the present disclosure.Pharmaceutical Food Supplement composition composition Fatty Acid K85EE,K85TG or Commercial up- Oil Mixture AGP103 drug substance concentratedoil mixture in EE and/or TG form Surfactant Tween ®20 or Tween ®40Tween ®20 or Tween ®40 Free Fatty (EPA-FAand DHA-FA), (EPA-FA + DHA-FA),Acid EPA-FA or DHA-FA EPA-FA or DHA-FA Total Oil 100% by weight 100% byweight Mixture 100% by weight

Further for example, K85EE omega-3 fatty acid oil and the free fattyacid chosen from K85FA having a EPA:DHA-FA ratio more or less equal tothe EPA: DHA-EE ratio in K85EE are exemplified in Table 26.

TABLE 26 Additional compositions according to the present disclosure.Total oil mixture content Free Fatty [oil:co-surfactant Fatty Acid FreeFatty Acid: EPA and Total oil ratio] in SMEDDS/ Oil Mixture: Free FattyAcid: EPA-FA DHA mixture mixture (by SEDDS Formulations K85EE Acid:K85-FA or DHA-FA in FA form weight) 1.) 80-95% 5-20 w % 100 w % 2.)70-80% 20-30% 100 w % 3.) 50-70% 30-50% 100 w % 4.) 50-60% 40-50% 100 w% 5.) 60-70% 30-40% 100 w % 6.) 70-80% 20-30% 100 w % 7.) 80-95%  5-20%100 w % 8.)  >80%  <20% 100 w % 9.) 70-80% 20-30% 100 w % 10.) 60-70%30-40% 100 w % 11.) 50-60% 40-50% 100 w % 12.) 85-95%  5-15% 100 w %EPA > DHA 13.) 80-90% 10-20% 100 w % EPA > DHA 14.) 70-80% 20-30% 100 w% EPA > DHA 15.) 60-70% 30-40% 100 w % EPA > DHA

Additionally, the total oil mixtures presented above can be mixed withthe surfactant Tween®20.

Further for example, the K85EE mixed fatty acid composition comprises atleast 90% omega-3 ethyl ester fatty acids, and wherein the mixed fattyacid composition comprises from about 80% to about 88% eicosapentaenoicacid ethyl ester and docosahexaenoic acid ethyl ester, by weight of thefatty acid composition.

A collection of ratios between [oil]:[surfactant]:[free fatty acid](a):b):c)) are illustrated in the table 30. For example, a K85EE orAGP103 oil is used together with a surfactant and a co-surfactant in the[K85EE]:[surfactant]:[free fatty acid] ranges from about 4:2:0.5 to4:4:2. Thus, the range for the surfactant may be from 2 to 4 and thefree fatty acid from 0.5 to 2.

It is also included herein that the K85EE oil mixture presented in Table27 above can be replaced by a K85TG oil mixture as well as a commercialomega-3 oil concentrate in ethyl ester and/or triglyceride form.

TABLE 27 SMEDDS formulations with Tween20, K85EE, EPA-FA or DHA-FA. EPA-200 mg K85EE Tween20 FA DHA-FA ~K85FA preconcentrate (mg) (mg) (mg) (mg)(mg) in 10 ml water A 400 400 100 emulsion B 400 400 100 emulsion C 400300 100 emulsion D 400 300 100 emulsion

Example 10: Pharmaceutical Preconcentrate Composition

A pharmaceutical preconcentrate composition was prepared by mixing thefollowing components:

as the fatty acid oil mixture: K85-EE; in an amount of 10.80 g;

as the surfactant: Tween-20 (Molecular Biology Grade, AppliChemDarmstadt, A4974,0250 lot 5N004174) in an amount of 7.44 g;

as the at least one fatty acid: EPA-FA in an amount of 1.53 g; andDHA-FA in an amount of 1.24 g.

With mixing, a transparent homogenous solution was obtained. The densityof the formulation was determined to be 1.02 g/ml. The composition wasthen filled in vials (vial seize=4 ml) each comprising 1.25×1670 mg=2087mg were prepared, flushed with nitrogen and sealed with parafilm.

Example 11: In Vivo Studies in Mini-Pig

Two different formulations were prepared and sent for in-vivo testing.Formulation 1 was prepared according to Example 12 above by mixing thefollowing components: K85EE, Tween20 EPA-FA and DHA-FA in the specifiedamounts, and Formulation 2 was OMACOR gelatine capsules.

The study was performed in 8 male Gottingen SPF minipigs from EllegaardGottingen Minipigs ApS. The animals were housed individually in floorpens (1.2 m²) with sawdust (“Jeluxyl” from Jelu Werk GmbH, Josef EhrlerGmbH & Co KG, Ludwigsmühle, D-73494 Rosenberg, Germany) as bedding.

Treatment was performed in a cross-over design. The dose was 2 g peranimal. The first day of treatment is designated Day 1. Treatment wasperformed with a wash out period of at least 10 days between eachdosing. Blood samples (n=8) were taken post-dosing. Plasma samples wereanalysed within 2 weeks for total lipid content of EPA and DHA by avalidated LC-MS/MS method. The result presented in FIG. 37 shows theplasma concentration versus time profile of the total lipidconcentration of EPA, supporting supra- bioavailability (e.g., greatthan 40%) for the K85 SMEDDS formulation. A similar results has alsobeen shown for the time profile of total lipid concentration of DHA (notshown in FIG. 37).

What is claimed is:
 1. A method of regulating at least one healthproblem in a subject in need thereof comprising administering to thesubject a supplement composition comprising: a fatty acid oil mixturecomprising from about 25% to about 75% eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA), by weight of the fatty acid oil mixture,wherein the EPA and DHA are in a form chosen from ethyl ester andtriglyceride; at least one free fatty acid chosen from EPA, DHA, ALA,HPA, DPA, ETA, ETE, STA, linoleic acid, GLA, AA, osbond acid, oleicacid, ricinoleic acid, erucic acid, and mixtures thereof; and at leastone surfactant; wherein the at least one health problem is chosen fromirregular plasma lipid levels, cardiovascular functions, immunefunctions, visual functions, insulin action, neuronal development, heartfailure, and post myocardial infarction.
 2. The method according toclaim 1, wherein the fatty acid oil mixture comprises from about 35% toabout 75% EPA and DHA, by weight of the fatty acid oil mixture.
 3. Themethod according to claim 1, wherein the fatty acid oil mixturecomprises from about 40% to about 70% EPA and DHA, by weight of thefatty acid oil mixture.
 4. The method according to claim 1, wherein thefatty acid oil mixture comprises from about 40% to about 60% EPA andDHA, by weight of the fatty acid oil mixture.
 5. The method according toclaim 1, wherein the fatty acid oil mixture comprises from about 40% toabout 55% EPA and DHA, by weight of the fatty acid oil mixture.
 6. Themethod according to claim 1, wherein the fatty acid oil mixturecomprises from about 50% to about 55% EPA and DHA, by weight of thefatty acid oil mixture.
 7. The method according to claim 1, wherein thefatty acid oil mixture is derived from at least one oil chosen frommarine oil, algae oil, plant-based oil, and microbial oil.
 8. The methodaccording to claim 7, wherein the marine oil is a purified fish oil. 9.The method according to claim 1, wherein the EPA:DHA weight ratio of thefatty acid oil mixture ranges from about 1:10 to 10:1.
 10. The methodaccording to claim 1, wherein the EPA:DHA weight ratio of the fatty acidoil mixture ranges from about 1:8 to 8:1.
 11. The method according toclaim 1, wherein the EPA:DHA weight ratio of the fatty acid oil mixtureranges from about 1:6 to 6:1.
 12. The method according to claim 1,wherein the EPA:DHA weight ratio of the fatty acid oil mixture rangesfrom about 1:5 to 5:1.
 13. The method according to claim 1, wherein theEPA:DHA weight ratio of the fatty acid oil mixture ranges from about 1:4to 4:1.
 14. The method according to claim 1, wherein the EPA:DHA weightratio of the fatty acid oil mixture ranges from about 1:3 to 3:1. 15.The method according to claim 1, wherein the EPA:DHA weight ratio of thefatty acid oil mixture ranges from about 1:2 to 2:1.
 16. The methodaccording to claim 1, wherein the EPA:DHA weight ratio of the fatty acidoil mixture ranges from about 1:1 to 2:1.
 17. The method according toclaim 1, wherein the EPA:DHA weight ratio of the fatty acid oil mixtureranges from about 1:2 to 1:3.
 18. The method according to claim 1,wherein the composition further comprises at least one antioxidant. 19.The method according to claim 1, wherein the composition furthercomprises at least one superdisintegrant chosen from crosscarmelose,crospovidone, and sodium starch glycolate, and wherein the compositionis in a tablet form.
 20. The method according to claim 19, wherein thecomposition comprises from about 1% to about 20% of the at least onesuperdisintegrant, by weight relative to the total weight of thecomposition.
 21. The method according to claim 1, wherein thecomposition is in the form of a gelatin capsule.
 22. The methodaccording to claim 21, wherein the capsule fill content ranges fromabout 0.400 g to about 1.300 g.
 23. The method according to claim 21,wherein the capsule fill content ranges from about 0.600 g to about1.200 g.
 24. The method according to claim 21, wherein the capsule fillcontent ranges from about 0.800 g to about 1.000 g.
 25. The methodaccording to claim 1, wherein the at least one surfactant is chosen fromanionic surfactants, nonionic surfactants, cationic surfactants,zwitterionic surfactants, and mixtures thereof, and a phospholipid,derivative thereof, analogue thereof, or any mixture thereof, to form asupplement preconcentrate.
 26. The method according to claim 25, whereinthe anionic surfactants are chosen from salts of perfluorocarboxylicacids and perfluorosulphonic acid, alkyl sulphate salts, sulphateethers, alkyl benzene sulphonate salts, and mixtures thereof.
 27. Themethod according to claim 25, wherein the nonionic surfactants arechosen from diacetyl monoglycerides, diethylene glycolmonopalmitostearates, ethylene glycol monopalmitostearates, glycerylbehenates, glyceryl distearates, glyceryl monolinoleates, glycerylmono-oleates, glyceryl monostearates, macrogol cetostearyl ethers,macrogol 15 hydroxystearates, macrogol lauril ethers, macrogolmonomethyl ethers, macrogol oleyl ethers, macrogol stearates, menfegol,mono and diglycerides, nonoxinols, octoxinols, polyoxamers, polyoxamer188, polyoxamer 407, polyoxyl castor oils, polyoxyl hydrogenated castoroils, propylene glycol diacetates, propylene glycol laureates, propyleneglycol monopalmitostearates, quillaia, sorbitan esters, sucrose esters,and mixtures thereof, and nonionic copolymers comprised of a centralhydrophobic polymer of polyoxypropylene(poly(propylene oxide)) with ahydrophilic polymer of at least one of polyethylene(poly(ethyleneoxide)), polyethylene ethers, sorbitan esters, polyoxyethylene fattyacid esters, polyethylated castor oil, and mixtures thereof.
 28. Themethod according to claim 27, wherein the nonionic surfactants arechosen from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate80, and mixtures thereof.
 29. The method according to claim 25, whereinthe cationic surfactants are chosen from quaternary ammonium compounds,cetylpyridinium chlorides, benzethonium chlorides, cetyltrimethylammonium bromides, and mixtures thereof.
 30. The methodaccording to claim 25, wherein the zwitterionic surfactants are chosenfrom dodecyl betaines, coco amphoglycinates, cocamidopropyl betaines,and mixtures thereof.
 31. The method according to claim 25, wherein thephospholipid, derivative thereof, or analogue thereof is chosen fromphosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols,phosphatidylserines, phosphatidylinositols, and mixtures thereof. 32.The method according to claim 25, wherein the composition furthercomprises at least one pharmaceutically-acceptable solvent chosen fromlower alcohols and polyols.
 33. The method according to claim 25,wherein the preconcentrate forms a self-nanoemulsifying drug deliverysystem (SNEDDS), self-microemulsifying drug delivery system (SMEDDS), orself-emulsifying drug delivery system (SEDDS) in an aqueous solution.